Diagnostic methods for neural disorders

ABSTRACT

The invention generally relates to optical methods for the diagnosis of neuronal condition by converting a cell from a patient into a neuron and optically evaluating action potentials of that cell in vitro. The cell is transformed with an optical reporter and exhibits an optical signature in response to neural stimulation. Using genome-editing, a control cell can be made that is isogenic but-for a known mutation and a control signature obtained from the control cell. Thus, methods of the invention reveal potential neurodegenerative effects of a mutation as manifested in a patient&#39;s genetic context. The optical signature of the cell, or the difference between the signature and the control signature, is correlated to a diagnosis of the neurodegenerative disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of, and priority to, U.S.Provisional Application Ser. No. 61/982,589, filed Apr. 22, 2014, thecontents of which are incorporated by reference.

FIELD OF THE INVENTION

The invention relates to optical methods for the diagnosis of diseaseaffecting neurons.

BACKGROUND

Neuronal diseases can be debilitating conditions that involve themalfunction, deterioration, or death of neurons. For example, when aperson suffers from a neurodegenerative disease, his or her neuronsdeteriorate, which can initially manifest as forgetfulness, cognitiveimpairment, or loss of coordination. As the disease progresses, theperson's condition can worsen considerably and he or she may becomeunable to walk and may suffer from severe dementia. Neurodegenerativediseases often present no outwardly visible symptoms until after theyhave caused significant harm to the nervous system.

Some neurodegenerative diseases are known to be associated with certainmutations or combinations of mutations. For example, variants of genessuch as C9orf72, SOD1, TARDBP, FUS, UBQL2, ALS2, and SETX are known tobe associated with amyotrophic lateral sclerosis or other neuraldisorders and the progression of the disease can vary depending on whatcombination of variants are present in a patient's genome. See Finsterer& Burgunder, 2014, Recent progress in the genetics of motor neurondisease, Eur J Med Genet, In press. As a consequence, simply by knowingthat a person has one disease-associated mutation (e.g., C9orf72), aclinician cannot conclude that a disorder will manifest in that person.Furthermore, distinct underlying causes of a disease, for instance dueto different mutations or due to differences in genetic background, maylead to outwardly similar sets of symptoms. Nonetheless, the treatmentmay need to be tailored to the underlying root-cause of the disease andto the particularities of each patient's genetic background.

Two factors conspire to prevent patient classification based solely upongenetic information: First, due to the vast number of possibledisease-causing mutations, many such mutations occur at a very low levelin the population. Additionally, there is a high level of geneticvariation in the population that is not directly associated withdisease. Thus from sequence alone, a clinician may not be able todetermine which mutations are causative in a disease. Even if themutation is found, the number of comparable cases may be so small thatdata on optimal treatment strategies is lacking.

SUMMARY

The invention provides methods for diagnosis of neuronal diseases byconverting a cell from a patient into a neuron and optically evaluatingaction potentials of that cell in vitro. A somatic cell is obtained froma patient and converted into a motor neuron or other cell type ofinterest. The neural cell is transformed with a genetically encodedoptical reporter, such as a transmembrane protein that fluoresces inresponse to the generation of an action potential. The cell, by theoptical reporter, exhibits an optical signature in response to neuralstimulation and that signature is observed and compared to a controlsignature, such as may be observed from a control cell with knownproperties. Differences between the observed signature and the controlsignature reveal properties of the patient cell and can be correlated toa diagnosis of a neurodegenerative disease. The invention uses methodsof converting somatic cells such as fibroblasts to specific neuralsubtypes as well as transformation of cells with optogenetic actuatorsand reporters to allow for characterizing cells optically. Imagescaptured by microscopy are analyzed digitally to identify opticalsignatures such as spike trains and associate the signatures withspecific cells. Disease-affected and healthy patient cells can bedistinguished according to their signature spike trains.

Using genome-editing, a practitioner can create patient-specific controlcells that are isogenic but-for specific genetic variants that aresuspected to be associated with disease. By these means, where a patientis known to have a certain mutation, methods of the invention can beused to see the consequences of that mutation within the genetic contextof the patient's entire genome. The effects of not just a singleidentified variant, but of that variant in the context of all otheralleles in the genome can be studied. Thus where a patient is known orsuspected of having a disease-associated mutation, methods of theinvention reveal potential neurodegenerative effects of that mutation asmanifested in that patient's genetic context, giving a clinician avaluable tool for diagnosis or treating a disease.

The presented methods are minimally invasive and can be performed forpatients of any age. Since the methods described here can be performedat an early age to diagnose a neurodegenerative disease, a disease canbe identified well before it has advanced significantly and causedsubstantial damage, which may allow medical science a better chance totreat the disease.

Aspects of the invention provide a method of diagnosing a condition. Thecondition may be any disease or disorder that involves or affectsneurons including developmental and genetic disorders andneurodegenerative diseases. A cell or cells are obtained from a personsuspected of having the condition. For example, the cell may be obtainedas a somatic cell (e.g., by dermal biopsy) from a patient. The cell ispreferably converted into a neuron or a specific neural sub-type such asa motor neuron. The cells are caused to express an optical reporter ofneural activity. The method includes observing a signature generated bythe optical reporter in response to a stimulation of the cell andcomparing the observed signature to a control signature. A differencebetween the observed signature and the control signature corresponds toa positive diagnosis of the condition. (In embodiments where the controlsignature is disease-type, a match between the observed signature andthe control signature corresponds to a positive diagnosis of thecondition.) The control signature may be obtained by obtaining a controlcell suspected of not having the condition and observing a controlsignal generated by a control optical reporter in the control cell. In apreferred embodiment, the control cell is derived from the test cell orcells that are changed by genomic editing. Obtaining the control cellmay include editing a genome from the subject such that the control celland the cell are isogenic but for a mutation. Alternatively, the controlcells may be derived from one or more individuals known not to have thecondition nor to have genetic mutations associated with risk of thecondition.

Any suitable condition may be diagnosed using the described methods.Methods of the invention are suited to diagnosing conditions such asgenetic disorders, mental and psychiatric conditions, neurodevelopmentaldisorders and neurodegenerative diseases. Exemplary genetic disordersinclude Cockayne syndrome, Down Syndrome, Dravet syndrome, familialdysautonomia, Fragile X Syndrome, Friedreich's ataxia, Gaucher disease,giant axonal neuropathy, Charcot-Marie-Tooth disease, hereditary spasticparaplegias, Machado-Joseph disease (also called spinocerebellar ataxiatype 3), Phelan-McDermid syndrome (PMDS), polyglutamine (polyQ)-encodingCAG repeats, a variety of ataxias including spinocerebellar ataxias,spinal muscular atrophy, and Timothy syndrome. Exemplaryneurodegenerative diseases include Alzheimer's disease, frontotemporallobar degeneration, Huntington's disease, multiple sclerosis,Parkinson's disease, spinal and bulbar muscular atrophy, and amyotrophiclateral sclerosis. Exemplary mental and psychiatric conditions includeschizophrenia. Exemplary neurodevelopmental disorders include Rettsyndrome. In one exemplary embodiment, the condition is amyotrophiclateral sclerosis (ALS). The patient may be known to have anALS-associated mutation, such as a mutation in a gene such as SOD1,TARDBP, FUS, UBQL2, ALS2, or SETX. In certain embodiments, the subjecthas a mutation in a SOD1 gene, such as the SOD1A4V mutation.

In some embodiments, the cell is caused to express an optical actuatorthat initiates an action potential in response to optical stimulation.Stimulation of the cell may include illuminating the optical actuator.

Causing the cell to express the optical reporter may be done bytransforming the cell with a vector bearing a genetically encodedfluorescent voltage reporter. The vector may also include a geneticallyencoded optical voltage actuator, such as a light-gated ion channel.

Observing the signal can include observing a cluster of different cellswith a microscope and using a computer to isolate the signal generatedby the optical reporter from a plurality of signals from the differentcells. Methods of the invention may include using the computer toisolate the signal by performing an independent component analysis orother source-separation algorithm. The computer may be used to identifya spike train associated with the cell using standard spike-findingalgorithms that apply steps of filtering the data and then applying athreshold. The computer may also be used to map propagation ofelectrical spikes within a single cell by means of an analyticalalgorithm such as a sub-Nyquist action potential timing algorithm.Methods may include observing and analyzing a difference between theobserved signal and the expected signal. The difference may manifest asa decreased or increased probability of a voltage spike in response tothe stimulation of the cell relative to a control, a change in thepropagation of the signal within a cell, a change in the transformationof the signal upon synaptic transmission, or a change in the waveform ofthe action potential.

In certain aspects, the invention provides compound screening methodthat includes converting a somatic cell to an electrically active cell,incorporating into the electrically active cell an optical activator andan optical reporter of electrical activity, and exposing the cells to atleast one compound. A signatures generated by the optical reporter inresponse to an optical stimulation of the cells is obtained and themethod includes identifying an effect of the at least one compound oncellular phenotype based on the obtained signature. Preferably, theelectrically active cell is a neuron, cardiomyocyte, or glial cell.“Electrically active cell” may be taken to refer to cells that transmita signal or an action potential or participate in neural or cardiacfunction and include neurons, cardiomyocytes, and glia. A plurality ofthe electrically active cells may be exposed to a plurality of differentcompounds. Any effect may be identified such as an effect thatrepresents cellular activity (action potential level, energy level,synaptic transmission).

In some embodiments, the somatic cell is obtained from a population ofdiseased cells. The method may include identifying the effectiveness ofthe compounds treating said diseased cells. Any disease may be modeledsuch as Cockayne syndrome, Down Syndrome, Dravet syndrome, familialdysautonomia, Fragile X Syndrome, Friedreich's ataxia, Gaucher disease,hereditary spastic paraplegias, Machado-Joseph disease, Phelan-McDermidsyndrome (PMDS), polyglutamine (polyQ)-encoding CAG repeats, spinalmuscular atrophy, Timothy syndrome, Alzheimer's disease, frontotemporallobar degeneration, Huntington's disease, multiple sclerosis,Parkinson's disease, spinal and bulbar muscular atrophy, and amyotrophiclateral sclerosis.

The converting step may proceed by direct lineage conversion orconversion through an iPS intermediary.

The incorporating may include transforming the electrically active cellswith a vector that includes a nucleic acid encoding the opticalactivator and the optical reporter of electrical activity. An opticalactivator may initiate an action potential in response to the opticalstimulation. The cells may be stimulated by illumination. In certainembodiments, each of the electrically active cell is caused to expressboth the optical activator and the optical reporter of electricalactivity.

The effect of the compound may be identified by comparing an electricalsignature to a control signature obtained from a control cell. Themethod may include editing the genome of the electrically active cellsto produce control cells such that the control cells and theelectrically active cells are isogenic but for a mutation in theelectrically active cells.

In some embodiments, the signature is obtained by observing a cluster ofcells with a microscope and using a computer to isolate a signalgenerated by the optical reporter from among a plurality of signals fromthe cluster of cells. An image can be obtained of a plurality ofclusters of cells using the microscope (i.e., all in a single imageusing a microscope of the invention). The computer isolates the signalby performing an independent component analysis and identifying a spiketrain produced by one single cell.

In certain aspects, the invention provides a method of treating acondition by obtaining a neuron derived from a somatic cell from aperson having the condition, incorporating into the neuron an opticalreporter of neural activity, and exposing the neuron to a candidatetreatment compound. A signature generated by the optical reporter inresponse to a stimulation of the cell is used to observe an influence ofthe compound on a phenotype of the cell and—where the compound isobserved to promote a normal-type phenotype—the compound is selected fortreating the patient. The condition may be, for example, Cockaynesyndrome, Down Syndrome, Dravet syndrome, familial dysautonomia, FragileX Syndrome, Friedreich's ataxia, Gaucher disease, hereditary spasticparaplegias, Machado-Joseph disease, Phelan-McDermid syndrome (PMDS),polyglutamine (polyQ)-encoding CAG repeats, spinal muscular atrophy,Timothy syndrome, Alzheimer's disease, frontotemporal lobardegeneration, Huntington's disease, multiple sclerosis, Parkinson'sdisease, spinal and bulbar muscular atrophy, or amyotrophic lateralsclerosis. Methods include causing the cell to express an opticalactuator that initiates an action potential in response to opticalstimulation. The cell may be stimulated by illuminating the opticalactuator. The cell may be obtained by obtaining a somatic cell from thesubject and converting the somatic cell into an electrically active celltype. In certain embodiments, the somatic cell is converted to a neuronand may be converted to a specific neural sub-type. The condition may beneuronal disorder such as a neurodegenerative disease. Conversion mayinclude direct lineage conversion or conversion through an iPSintermediary.

Observing the influence may include comparing the signature to a controlsignature obtained from a control cell, and the method further includesobtaining the control cell by editing a genome from the subject suchthat the control cell and the cell are isogenic but for a mutation. Theneuron may be transformed with a vector bearing a genetically encodedfluorescent voltage reporter, a genetically encoded optical voltageactuator, or both.

To observe the signal, a cluster of cells may be observed with amicroscope and a computer may isolate the signal generated by theoptical reporter from a plurality of signals from the different cells.In some embodiments, the computer isolates the signal by performing anindependent component analysis and identifying a spike train associatedwith the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams a method for diagnosing a condition.

FIG. 2 illustrates exemplary pathways for converting cells into specificneural subtypes.

FIG. 3 gives an overview of zinc-finger nuclease mediated editing.

FIG. 4 presents a structural model of an optical reporter of neuralactivity.

FIG. 5 diagrams components of an optical imaging apparatus.

FIG. 6 illustrates the use of pulse sequences to record actionpotentials.

FIG. 7 is an image of cells from which an individual is to be isolated.

FIG. 8 illustrates the isolation of individual cells in a field of view.

FIG. 9 shows the spike trains associated with individual cells.

FIG. 10 shows individual cells in a cluster color-coded after isolation.

FIG. 11 shows optical excitation being used to induce action potentials.

FIG. 12 shows eigenvectors from a principal component analysis (PCA).

FIG. 13 shows a relation between cumulative variance and eigenvectornumber.

FIG. 14 gives a comparison of action potential waveforms.

FIG. 15 shows an action potential timing map.

FIG. 16 shows the accuracy of timing extracted by methods of theinvention.

FIG. 17 gives an image of fluorescence distribution of an opticalactuator.

FIG. 18 presents frames from a SNAPT movie.

FIG. 19 compares spike probability of wild-type and mutant cells.

FIG. 20 presents a system useful for performing methods of theinvention.

FIG. 21 gives a comparison of AP waveforms as measured by thegenetically encoded voltage indicator QuasAr2 and the voltage-sensitivedye, FluoVolt.

FIG. 22 shows plots of the average waveforms from the traces in FIG. 21.

FIG. 23 presents phototoxicity and photobleaching measurement ofQuasAr2.

FIG. 24 graphs the average AP waveform shapes.

FIG. 25 shows optogenetic proteins used for stimulus and detection ofvoltage and intracellular Ca2+.

FIG. 26 illustrates cellular plating configurations.

FIG. 27 shows cells expressing CheRiff plated in an annular region.

DETAILED DESCRIPTION

The invention provides methods for the optical diagnosis of diseasesaffecting electrically active cells. Methods may be used to diagnosediseases affecting neurons or cardiomyocytes, for example. In someembodiments, methods of the invention are used to diagnoses a conditionknown to be associated with a genetic variant, or mutation.

FIG. 1 diagrams a method 101 for diagnosing a condition according toembodiments of the invention. This may involve obtaining 107 a cell froma person suspected of having the condition. Genome editing techniques(e.g., use of transcription activator-like effector nucleases (TALENs),the CRISPR/Cas system, zinc finger domains) may be used to create acontrol cell that is isogenic but-for a variant of interest. The celland the control are converted into an electrically excitable cell suchas a neuron, astrocyte, or cardiomyocyte. The cell may be converted to aspecific neural subtype (e.g., motor neuron). The cells are caused toexpress 113 an optical reporter of neural activity. For example, thecell may be transformed with a vector comprising an optogenetic reporterand the cell may also be caused to express an optogenetic actuator (akaactivator) by transformation. Optionally, a control cell may beobtained, e.g., by taking another sample, by genome editing, or by othersuitable techniques. Using microscopy and analytical methods describedherein, the cells are observed and specifically, the cells' response tostimulation 119 (e.g., optical, synaptic, chemical, or electricalactuation) may be observed. A cell's characteristic signature such as aneural response as revealed by a spike train may be observed 123. Theobserved signature is compared to a control signature and a difference(or match) between the observed signature and the control signaturecorresponds to a positive diagnosis of the condition.

In one exemplary embodiment discussed herein, methods of the inventionare used for optical differentiation of amyotrophic lateral sclerosis(ALS) arising from a monogenic mutation in the SOD1 gene (SOD1A4V).

1. Obtaining Cell(s)

Cells are obtained from a person suspected of having the condition. Anysuitable condition such as a genetic disorder, mental or psychiatriccondition, neurodegenerative disease or neurodevelopmental disorder, orcardiac condition may be diagnosed. Additionally, methods of theinvention and the analytical pipelines described herein may be appliedto any condition for which an electrophysiological phenotype has beendeveloped. Exemplary genetic disorders suitable for analysis by apipeline defined by methods of the invention include Cockayne syndrome,Down Syndrome, Dravet syndrome, familial dysautonomia, Fragile XSyndrome, Friedreich's ataxia, Gaucher disease, hereditary spasticparaplegias, Machado-Joseph disease (also called spinocerebellar ataxiatype 3), Phelan-McDermid syndrome (PMDS), polyglutamine (polyQ)-encodingCAG repeats, giant axonal neuropathy, Charcot-Marie-Tooth disease, avariety of ataxias including spinocerebellar ataxias, spinal muscularatrophy, and Timothy syndrome. Exemplary neurodegenerative diseasesinclude Alzheimer's disease, frontotemporal lobar degeneration,Huntington's disease, multiple sclerosis, Parkinson's disease, spinaland bulbar muscular atrophy, and amyotrophic lateral sclerosis.Exemplary mental and psychiatric conditions include schizophrenia.Exemplary neurodevelopmental disorders include Rett syndrome. Whilediscussed here in terms of neuronal disorders, it will be appreciatedthat the methods described herein may be extended to the diagnosis ofcardiac disorders and cells may be converted to cardiomyocytes.Exemplary cardiac conditions include long-QT syndromes, hypertrophiccardiomyopathies, and dilated cardiomyopathies. Moreover,electrophysiological phenotypes for a variety of conditions have beendeveloped and reported in the literature.

Methods of the invention may include obtaining at least one neuron thathas a genotype or phenotype associated with autism, such as a cell witha genome having a mutation in a gene linked to autism. Mutations in anumber of genes have been linked to the development of autism, includingSHANK3 (ProSAP2), CDH9, CDH10, MAPK3, SERT (SLC6A4), CACNA1G, GABRB3,GABRA4, EN2, the 3q25-27 locus, SLC25A12, HOXA1, HOXA2, PRKCB1, MECP2,UBE3A, NLGN3, MET, CNTNAP2, FOXP2, GSTP1, PRL, PRLR, and OXTR. Genessuch as the SHANK3 have been studied in mouse models through N-terminaland PDZ domain knock-outs which resulted in phenotypes includingimpaired social interaction. Wang, et al., 2011, Synaptic dysfunctionand abnormal behaviors in mice lacking major isoforms of Shank3, Hum.Mol. Genet. 20 (15): 3093-108; Bozdagi, et al., 2010, Haploinsufficiencyof the autism-associated Shank3 gene leads to deficits in synapticfunction, social interaction, and social communication, Mol Autism 1(1): 15; Peça, et al., 2011, Shank3 mutant mice display autistic-likebehaviours and striatal dysfunction, Nature 472 (7344): 437-42; each ofwhich is incorporated by reference.

Dravet syndrome, also known as Severe Myoclonic Epilepsy of Infancy(SMEI), is a form of intractable epilepsy that begins in infancy and isoften associated with mutations in the SCN1A gene or certain other genessuch as SCN9A, SCN2B, PCDH19 or GABRG2. Dravet syndrome is discussed inHigurashi et al., 2013, A human Dravet syndrome model from patientinduced pluripotent stem cells, Mol Brain 6:19, the contents of whichare incorporated by reference. Other forms of epilepsy includegeneralized epilepsy with febrile seizures plus (GEFS+) which is thoughtto include Dravet syndrome, borderline severe myoclonic epilepsy ofinfancy (SMEB), and intractable epilepsy of childhood (IEC). Additionalneurodevelopmental disorders associated with epilepsy which may bestudied with the cells and methods of the invention include Angelmansyndrome, Rolandic epilepsy, autosomal dominant nocturnal frontal lobeepilepsy, benign occipital epilepsies of childhood, Panalyiotopoulossyndrome, childhood absence epilepsy, epilepsy-intellectual disabilityin females, febrile lobe epilepsy, juvenile myoclonic epilepsy,Lennox-Gastaut syndrome, Ohtahara syndrome, photosensitive epilepsy,pyridoxine-dependent epilepsy, Unverricht-Lundborg disease, myoclonicepilepsy with ragged red fibers syndrome, Lafora disease, Rasmussen'sencephalitis, ring chromosome 20 syndrome, temporal lobe epilepsy,tuberous sclerosis, and West syndrome. Additional genes associated withepilepsy which may be studied with the cells and methods of theinvention include, WWOX, PRRT2, KCNC1, STX1B, CARS2, STXB1, KCNQ2,CDKL5, ARX, SPTAN, BRAT1, KCNQ3, SCN2A (NAV1.2), GABA receptors, NIPA2,CDKL5, PCDH19, and NAV1.1.

Tuberous sclerosis is a genetic disease that affects tumor suppressorproteins through mutations to the TSC1 or TSC2 genes. Tuberous sclerosiscan result in tumor growth in the brain, kidneys, lungs, heart, skin,eyes and can negatively affect function of these organs. Neurologicalsymptoms of tuberous sclerosis include autism, intellectualdisabilities, developmental and behavioral problems, and seizures.People suffering from tuberous sclerosis face a range of prognoses basedon the severity of their symptoms, ranging from mild skin abnormalitiesto severe mental disabilities and organ failure and death due to tumorgrowth. Tuberous sclerosis is discussed in Meikle, et al., 2007, A mousemodel of tuberous sclerosis: neuronal loss of Tscl causes dysplastic andectopic neurons, reduced myelination, seizure activity, and limitedsurvival, J Neurosci. 27(21):5546-58; Meikle, et al., 2008, Response ofa neuronal model of tuberous sclerosis to mammalian target of rapamycin(mTOR) inhibitors: effects on mTORC1 and Akt signaling lead to improvedsurvival and function, J Neurosci., 28(21):5422-32; Normand, et al.,2013, Temporal and mosaic Tscl deletion in the developing thalamusdisrupts thalamocortical circuitry, neural function, and behavior,Neuron, 5; 78(5):895-909; Kim, et al., 2010, Zebrafish model of tuberoussclerosis complex reveals cell-autonomous and non-cell-autonomousfunctions of mutant tuberin, Dis Model Mech., 4(2):255-67; andWlodarski, et al., 2008, Tuberin-heterozygous cell line TSC2ang1 as amodel for tuberous sclerosis-associated skin lesions, Int J Mol Med.21(2):245-50; each incorporated in its entirety.

Parkinson's disease is a neurodegenerative disorder of the centralnervous system that involves the death of dopamine-generating cells inthe substantia nigra in the midbrain. Parkinson's disease is discussedin Cooper et al., 2012, Pharmacological rescue of mitochondrial deficitsin iPSC-derived neural cells from patients with familial Parkinson'sdisease, Sci Transl Med 4(141):141ra90; Chung et al., 2013,Identification and rescue of α-synuclein toxicity in Parkinsonpatient-derived neurons, Science 342(6161):983-7; Seibler et al., 2011,Mitochondrial Parkin recruitment is impaired in neurons derived frommutant PINK1 induced pluripotent stem cells, J Neurosci 31(16):5970-6;Sanchez-Danes et al., 2012, Disease-specific phenotypes in dopamineneurons from human iPS-based models of genetic and sporadic Parkinson'sdisease, EMBO Mol Med 4(5):380-395; Sanders et al., 2013, LRRK2mutations cause mitochondrial DNA damage in iPSC-derived neural cellsfrom Parkinson's disease patients: reversal by gene correction.Neurobiol Dis 62:381-6; and Reinhardt et al., 2013, Genetic correctionof a LRRK2 mutation in human iPSCs links parkinsonian neurodegenerationto ERK-dependent changes in gene expression, Cell Stem Cell12(3):354-367; LRRK2 mutant iPSC-derived DA neurons demonstrateincreased susceptibility to oxidative stress, the contents of each ofwhich are incorporated by reference

Cockayne syndrome is a genetic disorder caused by mutations in the ERCC6and ERCC8 genes and characterized by growth failure, impaireddevelopment of the nervous system, photosensitivity, and prematureaging. Cockayne syndrome is discussed in Andrade et al., 2012, Evidencefor premature aging due to oxidative stress in iPSCs from Cockaynesyndrome, Hum Mol Genet 21:3825-3834, the contents of which areincorporated by reference.

Down syndrome is a genetic disorder caused by the presence of all orpart of a third copy of chromosome 21 and associated with delayedgrowth, characteristic facial features, and intellectual disability.Down Syndrome is discussed in Shi et al., 2012, A human stem cell modelof early Alzheimer's disease pathology in Down syndrome, Sci Transl Med4(124):124ra129, the contents of which are incorporated by reference.

Dravet syndrome, also known as Severe Myoclonic Epilepsy of Infancy(SMEI), is a form of intractable epilepsy that begins in infancy and isoften associated with mutations in the SCN1A gene or certain other genessuch as SCN9A, SCN2B, PCDH19 or GABRG2. Dravet syndrome is discussed inHigurashi et al., 2013, A human Dravet syndrome model from patientinduced pluripotent stem cells, Mol Brain 6:19, the contents of whichare incorporated by reference.

Familial dysautonomia is a genetic disorder of the autonomic nervoussystem caused by mutations in the IKBKAP gene and that affects thedevelopment and survival of sensory, sympathetic and someparasympathetic neurons in the autonomic and sensory nervous systemresulting in variable symptoms including: insensitivity to pain,inability to produce tears, poor growth, and labile blood pressure.Familial dysautonomia is discussed in Lee et al., 2009, Modellingpathogenesis and treatment of familial dysautonomia usingpatient-specific iPSCs, Nature 461:402-406, the contents of which areincorporated by reference.

Fragile X syndrome is a genetic condition caused by mutations in theFMR1 gene and that causes a range of developmental problems includinglearning disabilities and cognitive impairment. Fragile X Syndrome isdiscussed in Liu et al., 2012, Signaling defects in iPSC-derived fragileX premutation neurons, Hum Mol Genet 21:3795-3805, the contents of whichare incorporated by reference.

Friedreich ataxia is an autosomal recessive ataxia resulting from amutation of a gene locus on chromosome 9. The ataxia of Friedreich'sataxia results from the degeneration of nerve tissue in the spinal cord,in particular sensory neurons essential (through connections with thecerebellum) for directing muscle movement of the arms and legs. Thespinal cord becomes thinner and nerve cells lose some of their myelinsheath. Friedreich's ataxia is discussed in Ku et al., 2010,Friedreich's ataxia induced pluripotent stem cells modelintergenerational GAA.TTC triplet repeat instability, Cell Stem Cell7(5):631-7; Du et al., 2012, Role of mismatch repair enzymes in GAA.TTCtriplet-repeat expansion in Friedreich ataxia induced pluripotent stemcells, J Biol Chem 287(35):29861-29872; and Hick et al., 2013, Neuronsand cardiomyocytes derived from induced pluripotent stem cells as amodel for mitochondrial defects in Friedreich's ataxia, Dis Model Mech6(3):608-21, the contents of each of which are incorporated byreference.

Gaucher's disease is a genetic disease caused by a recessive mutation ina gene located on chromosome 1 and in which lipids accumulate in thebody. Gaucher disease is discussed in Mazzulli et al., 2011, Gaucherdisease glucocerebrosidase and α-synuclein form a bidirectionalpathogenic loop in synucleinopathies, Cell 146(1):37-52, the contents ofwhich are incorporated by reference.

Hereditary Spastic Paraplegia (HSP)—also called Familial SpasticParaplegias, French Settlement Disease, or Strumpell-Lorraindisease—refers to a group of inherited diseases characterized by axonaldegeneration and dysfunction resulting in stiffness and contraction(spasticity) in the lower limbs. Hereditary spastic paraplegias isdiscussed in Denton et al., 2014, Loss of spastin function results indisease-specific axonal defects in human pluripotent stem cell-basedmodels of hereditary spastic paraplegia, Stem Cells 32(2):414-23, thecontents of which are incorporated by reference.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Josephdisease, is a neurodegenerative disease, an autosomal dominantlyinherited ataxia characterized by the slow degeneration of thehindbrain. Machado-Joseph disease (also called spinocerebellar ataxiatype 3) is discussed in Koch et al., 2011, Excitation-induced ataxin-3aggregation in neurons from patients with Machado-Joseph disease, Nature480(7378):543-546, the contents of which are incorporated by reference.

Phelan-McDermid Syndrome (PMDS) is a progressive neurodevelopmentaldisorder resulting from mutations in or deletions of the neural protein,Shank3 and characterized by developmental delay, impaired speech, andautism. Phelan-McDermid syndrome (PMDS) is discussed in Shcheglovitov etal., 2013, SHANK3 and IGF1 restore synaptic deficits in neurons from22q13 deletion syndrome patients, Nature 503(7475):267-71, the contentsof which are incorporated by reference.

Trinucleotide repeat disorders are characterized by polyglutamine(polyQ)-encoding CAG repeats. Trinucleotide repeat disorders refer to aset of genetic disorders caused by trinucleotide repeat expansion, whichdisorders include dentatorubropallidoluysian atrophy, Huntington'sdisease, spinobulbar muscular atrophy, Spinocerebellar ataxia Type 1,Spinocerebellar ataxia Type 2, Spinocerebellar ataxia Type 3 orMachado-Joseph disease, Spinocerebellar ataxia Type 6, Spinocerebellarataxia Type 7, and Spinocerebellar ataxia Type 17, as well as a varietyof other ataxias. Trinucleotide repeat disorders are discussed in HDiPSC Consortium, 2012, Induced pluripotent stem cells from patients withHuntington's disease show CAG-repeat-expansion-associated phenotypes.Cell Stem Cell 11(2):264-278, the contents of which are incorporated byreference.

Giant axonal neuropathy is a neurological disorder that causesdisorganization of neurofilaments, which form a structural framework todefine the shape and size of neurons. Giant axonal neuropathy resultsfrom mutations in the GAN gene, which codes for the protein gigaxonin.See Mahammad et al., 2013, Giant axonal neuropathy-associated gigaxoninmutations impair intermediate filament protein degredation, J ClinInvest 123(5):1964-75.

Charcot Marie Tooth disease, also known as hereditary motor and sensoryneuropathy (HMSN) and peroneal muscular atrophy (PMA), refers to severalinherited disorders of the peripheral nervous system characterized byprogressive loss of muscle and sensation. See, e.g., Harel and Lupski,2014, Charcot Marie Tooth disease and pathways to molecular basedtherapies, Clin Genet DOI: 10.1111/cge.12393.

Spinal muscular atrophy (SMA) is genetic disease caused by mutations inthe SMN1 gene, which encodes the survival of motor neuron protein (SMN),the diminished abundance of which neurons results in death of neuronalcells in the spinal cord and system-wide atrophy. Spinal muscularatrophy is discussed in Ebert et al., 2009, Induced pluripotent stemcells from a spinal muscular atrophy patient, Nature 457(7227):277-80;Sareen et al., 2012, Inhibition of apoptosis blocks human motor neuroncell death in a stem cell model of spinal muscular atrophy. PLoS One7(6):e39113; and Corti et al., 2012, Genetic correction of human inducedpluripotent stem cells from patients with spinal muscular atrophy, SciTransl Med 4 (165):165ra162, the contents of each of which areincorporated by reference.

Timothy syndrome is a genetic disorder arising from a mutation in theCa(v)1.2 Calcium Channel gene called CACNA1C and characterized by aspectrum of problems that include an abnormally prolonged cardiac“repolarization” time (long QT interval) and other neurological anddevelopmental defects, including heart QT-prolongation, heartarrhythmias, structural heart defects, syndactyly and autism spectrumdisorders. Timothy syndrome is discussed in Krey et al., 2013, Timothysyndrome is associated with activity-dependent dendritic retraction inrodent and human neurons, Nat Neurosci 16(2):201-9, the contents ofwhich are incorporated by reference.

Mental and psychiatric disorders such as schizophrenia and autism mayinvolve cellular and molecular defects amenable to study via stem cellmodels and may be caused by or associated with certain geneticcomponents that can be isolated using methods herein. Schizophrenia isdiscussed in Brennand et al., 2011, Modelling schizophrenia using humaninduced pluripotent stem cells, Nature 473(7346):221-225; and Chiang etal., 2011, Integration-free induced pluripotent stem cells derived fromschizophrenia patients with a DISC1 mutation, Molecular Psych16:358-360, the contents of each of which are incorporated by reference.

Alzheimer's disease is a neurodegenerative disease of uncertain cause(although mutations in certain genes have been linked to the disorder)and is one of the most common forms of dementia. Alzheimer's disease isdiscussed in Israel et al., 2012, Probing sporadic and familialAlzheimer's disease using induced pluripotent stem cells, Nature482(7384):216-20; Muratore et al., 2014, The familial Alzheimer'sdisease APPV717I mutation alters APP processing and tau expression iniPSC-derived neurons, Human Molecular Genetics, in press; Kondo et al.,2013, Modeling Alzheimer's disease with iPSCs reveals stress phenotypesassociated with intracellular Abeta and differential drugresponsiveness, Cell Stem Cell 12(4):487-496; and Shi et al., 2012, Ahuman stem cell model of early Alzheimer's disease pathology in Downsyndrome, Sci Transl Med 4(124):124ra129, the contents of each of whichare incorporated by reference.

Frontotemporal lobar degeneration (FTLD) is the name for a group ofclinically, pathologically and genetically heterogeneous disordersincluding frontotemporal dementia (which subdivides to includebehavioral-variant frontotemporal dementia (bvFTLD); semantic dementia(SD); and progressive nonfluent aphasia (PNFA)) associated with atrophyin the frontal lobe and temporal lobe of the brain. Frontotemporal lobardegeneration is discussed in Almeida et al, 2013, Modeling keypathological features of frontotemporal dementia with C9ORF72 repeatexpansion in iPSC-derived human neurons, Acta Neuropathol126(3):385-399; Almeida et al., 2012, Induced pluripotent stem cellmodels of progranulin-deficient frontotemporal dementia uncover specificreversible neuronal defects, Cell Rep 2(4):789-798; and in Fong et al.,2013, Genetic correction of tauopathy phenotypes in neurons derived fromhuman induced pluripotent stem cells, Stem Cell Reports 1(3):1-9, thecontents of each of which are incorporated by reference.

Huntington's disease is an inherited disease that causes the progressivedegeneration of nerve cells in the brain and is caused by an autosomaldominant mutation in either of an individual's two copies of a genecalled Huntingtin (HTT) located on the short arm of chromosome 4.Huntington's disease is discussed in HD iPSC Consortium, 2012, Inducedpluripotent stem cells from patients with Huntington's disease showCAG-repeat-expansion-associated phenotypes. Cell Stem Cell11(2):264-278; An et al., 2012, Genetic correction of Huntington'sdisease phenotypes in induced pluripotent stem cells, Cell Stem Cell11(2):253-263; and Camnasio et al., 2012, The first reported generationof several induced pluripotent stem cell lines from homozygous andheterozygous Huntington's disease patients demonstrates mutation relatedenhanced lysosomal activity, Neurobiol Dis 46(1):41-51, the contents ofeach of which are incorporated by reference.

Multiple sclerosis is a neurodegenerative disease in which theinsulating covers of nerve cells in the brain and spinal cord aredamaged. Multiple sclerosis is discussed in Song et al., 2012, Neuraldifferentiation of patient specific iPS cells as a novel approach tostudy the pathophysiology of multiple sclerosis, Stem Cell Res8(2):259-73, the contents of which are incorporated by reference.

Parkinson's disease is a neurodegenerative disorder of the centralnervous system that involves the death of dopamine-generating cells inthe substantia nigra in the midbrain. Parkinson's disease is discussedin Cooper et al., 2012, Pharmacological rescue of mitochondrial deficitsin iPSC-derived neural cells from patients with familial Parkinson'sdisease, Sci Transl Med 4(141):141ra90; Chung et al., 2013,Identification and rescue of α-synuclein toxicity in Parkinsonpatient-derived neurons, Science 342(6161):983-7; Seibler et al., 2011,Mitochondrial Parkin recruitment is impaired in neurons derived frommutant PINK1 induced pluripotent stem cells, J Neurosci 31(16):5970-6;Sanchez-Danes et al., 2012, Disease-specific phenotypes in dopamineneurons from human iPS-based models of genetic and sporadic Parkinson'sdisease, EMBO Mol Med 4(5):380-395; Sanders et al., 2013, LRRK2mutations cause mitochondrial DNA damage in iPSC-derived neural cellsfrom Parkinson's disease patients: reversal by gene correction.Neurobiol Dis 62:381-6; and Reinhardt et al., 2013, Genetic correctionof a LRRK2 mutation in human iPSCs links parkinsonian neurodegenerationto ERK-dependent changes in gene expression, Cell Stem Cell12(3):354-367; LRRK2 mutant iPSC-derived DA neurons demonstrateincreased susceptibility to oxidative stress, the contents of each ofwhich are incorporated by reference.

Spinal and bulbar muscular atrophy (SBMA), also known as spinobulbarmuscular atrophy, bulbo-spinal atrophy, X-linked bulbospinal neuropathy(XBSN), X-linked spinal muscular atrophy type 1 (SMAX1), and Kennedy'sdisease (KD)—is a neurodegenerative disease associated with mutation ofthe androgen receptor (AR) gene and that results in muscle cramps andprogressive weakness due to degeneration of motor neurons in the brainstem and spinal cord. Spinal and bulbar muscular atrophy is discussed inNihei et al., 2013, Enhanced aggregation of androgen receptor in inducedpluripotent stem cell-derived neurons from spinal and bulbar muscularatrophy, J Biol Chem 288(12):8043-52, the contents of which areincorporated by reference.

Rett syndrome is a neurodevelopmental disorder generally caused by amutation in the methyl CpG binding protein 2, or MECP2, gene and whichis characterized by normal early growth and development followed by aslowing of development, loss of purposeful use of the hands, distinctivehand movements, slowed brain and head growth, problems with walking,seizures, and intellectual disability. Rett syndrome is discussed inMarchetto et al., 2010, A model for neural development and treatment ofRett syndrome using human induced pluripotent stem cells, Cell,143(4):527-39 and in Ananiev et al., 2011, Isogenic pairs of wild typeand mutant induced pluripotent stem cell (iPSC) lines from Rett syndromepatients as in vitro disease model, PLoS One 6(9):e25255, the contentsof each of which are incorporated by reference.

In one illustrative example, the condition is amyotrophic lateralsclerosis. Amyotrophic lateral sclerosis (ALS), often referred to as“Lou Gehrig's Disease,” is a neurodegenerative disease associated withthe progressive degeneration and death of the motor neurons and aresultant loss of muscle control or paralysis. Amyotrophic lateralsclerosis is discussed in Kiskinis et al., 2014, Pathways disrupted inhuman ALS motor neurons identified through genetic correction of mutantSOD1, Cell Stem Cell (epub); Wainger et al., 2014, Intrinsic membranehyperexcitability of amyotrophic lateral sclerosis patient-derived motorneurons, Cell Reports 7(1):1-11; Donnelly et al., 2013, RNA toxicityfrom the ALS/FTD C9orf72 expansion is mitigated by antisenseintervention, Neuron 80(2):415-28; Alami, 2014, Microtubule-dependenttransport of TDP-43 mRNA granules in neurons is impaired by ALS-causingmutations, Neuron 81(3):536-543; Donnelly et al., 2013, RNA toxicityfrom the ALS/FTD C9ORF72 expansion is mitigated by antisenseintervention, Neuron 80(2):415-428; Bilican et al, 2012, Mutant inducedpluripotent stem cell lines recapitulate aspects of TDP-43proteinopathies and reveal cell-specific vulnerability, PNAS109(15):5803-5808; Egawa et al., 2012, Drug screening for ALS usingpatient-specific induced pluripotent stem cells, Sci Transl Med4(145):145ra104; and in Yang et al., 2013, A small molecule screen instem-cell-derived motor neurons identifies a kinase inhibitor as acandidate therapeutic for ALS, Cell Stem Cell 12(6):713-726, thecontents of each of which are incorporated by reference.

In one illustrative example, fibroblasts may be taken from a patientknown or suspected to have a mutation such as a mutation in SOD1. Anysuitable cell may be obtained and any suitable method of obtaining asample may be used. In some embodiments, a dermal biopsy is performed toobtain dermal fibroblasts. The patient's skin may be cleaned and givenan injection of local anesthetic. Once the skin is completelyanesthetized, a sterile 3 mm punch is used. The clinician may applypressure and use a “drilling” motion until the punch has pierced theepidermis. The punch will core a 3 mm cylinder of skin. The clinicianmay use forceps to lift the dermis of the cored skin and a scalpel tocut the core free. The biopsy sample may be transferred to a sterile BMEfibroblast medium after optional washing with PBS and evaporation of thePBS. The biopsy site on the patient is dressed (e.g., with an adhesivebandage). Suitable methods and devices for obtaining the cells arediscussed in U.S. Pat. Nos. 8,603,809; 8,403,160; 5,591,444; U.S. Pub.2012/0264623; and U.S. Pub. 2012/0214236, the contents of each of whichare incorporated by reference. Any tissue culture technique that issuitable for the obtaining and propagating biopsy specimens may be usedsuch as those discussed in Freshney, Ed., 1986, Animal Cell Culture: APractical Approach, IRL Press, Oxford England; and Freshney, Ed., 1987,Culture of Animal Cells: A Manual of Basic Techniques, Alan R. Liss &Co., New York, both incorporated by reference.

2. Converting Cell(s) into Neurons, Cardiomyocytes, or Specific NeuralSub-Types

Obtained cells may be converted into any electrically excitable cellssuch as neurons, specific neuronal subtypes, astrocytes or other glia,cardiomyocytes, or immune cells. Additionally, cells may be convertedand grown into co-cultures of multiple cell types (e.g. neurons+glia,neurons+cardiomyocytes, neurons+immune cells).

FIG. 2 illustrates exemplary pathways for converting cells into specificneural subtypes. A cell may be converted to a specific neural subtype(e.g., motor neuron). Suitable methods and pathways for the conversionof cells include pathway 209, conversion from somatic cells to inducedpluripotent stem cells (iPSCs) and conversion of iPSCs to specific celltypes, or pathways 211 direct conversion of cells in specific celltypes.

2a. Conversion of Cells to iPSs and Conversion of iPSs to Specific CellTypes

Following pathways 209, somatic cells may be reprogrammed into inducedpluripotent stem cells (iPSCs) using known methods such as the use ofdefined transcription factors. The iPSCs are characterized by theirability to proliferate indefinitely in culture while preserving theirdevelopmental potential to differentiate into derivatives of all threeembryonic germ layers. In certain embodiments, fibroblasts are convertedto iPSC by methods such as those discussed in Takahashi and Yamanaka,2006, Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors Cell 126:663-676.; and Takahashi,et al., 2007, Induction of pluripotent stem cells from adult humanfibroblasts by defined factors, Cell 131:861-872.

Induction of pluripotent stem cells from adult fibroblasts can be doneby methods that include introducing four factors, Oct3/4, Sox2, c-Myc,and Klf4, under ES cell culture conditions. Human dermal fibroblasts(HDF) are obtained. A retroviruses containing human Oct3/4, Sox2, Klf4,and c-Myc is introduced into the HDF. Six days after transduction, thecells are harvested by trypsinization and plated onto mitomycinC-treated SNL feeder cells. See, e.g., McMahon and Bradley, 1990, Cell62:1073-1085. About one day later, the medium (DMEM containing 10% FBS)is replaced with a primate ES cell culture medium supplemented with 4ng/mL basic fibroblast growth factor (bFGF). See Takahashi, et al.,2007, Cell 131:861. Later, hES cell-like colonies are picked andmechanically disaggregated into small clumps without enzymaticdigestion. Each cell should exhibit morphology similar to that of humanES cells, characterized by large nuclei and scant cytoplasm. The cellsafter transduction of HDF are human iPS cells. DNA fingerprinting,sequencing, or other such assays may be performed to verify that the iPScell lines are genetically matched to the donor.

These iPS cells can then be differentiated into specific neuronalsubtypes. Pluripotent cells such as iPS cells are by definition capableof differentiating into cell types characteristic of different embryonicgerm layers. A property of both embryonic stem cells human iPS cells istheir ability, when plated in suspension culture, to form embryoidbodies (EBs). EBs formed from iPS cells are treated with two smallmolecules: an agonist of the sonic hedgehog (SHH) signaling pathway andretinoic acid (RA). For more detail, see the methods described in Dimoset al., 2008, Induced pluripotent stem cells generated from patientswith ALS can be differentiated into motor neurons, Science321(5893):1218-21; Amoroso et al., 2013, Accelerated high-yieldgeneration of limb-innervating motor neurons from human stem cells, JNeurosci 33(2):574-86; and Boulting et al., 2011, A functionallycharacterized test set of human induced pluripotent stem cells, NatBiotech 29(3):279-286.

Aspects of the invention provide cellular disease models in which stemcells may be converted into functional neurons by forced expression of asingle transcription factor and then also caused to express optogeneticreporters or actuators of neural activity. A transcription factor suchas neurogenin-2 (NgN2) or NeurD1 introduced into a pluripotent stem cellby transfection is expressed, causing the cell to differentiate into aneuron. Additionally or separately an optogenetic construct thatincludes an optical reporter of intracellular calcium as well as anoptical actuator or reporter of membrane potential is expressed.

In some embodiments, conversion includes causing a stem cell to expressa single transcription factor. Overexpressing a single transcriptionfactor such as neurogenin-2 (Ngn2) or NeuroD1 alone rapidly converts ESand iPS cells into neuronal cells. See Zhang et al., 2013, Rapidsingle-step induction of functional neurons from human pluripotent stemcells, Neuron 78(5):785-798. The transcription factor may be introducedby lentiviral infection (discussed in greater detail below). As reportedin Zhang 2013 a puromycin resistance gene may be co-expressed with Ngn2for selection. ES or iPS cells are plated on day −2, infected withlentiviruses on day −1, and Ngn2 expression is induced on day 0. A 24 hrpuromycin selection period is started on day 1, and mouse glia(primarily astrocytes) are added on day 2 to enhance synapse formation.Forced Ngn2 expression converts ES and iPS cells into neuron-like cellsin less than one week, and produces an apparently mature neuronalmorphology in less than two weeks, as reported in Zhang 2013.

When the differentiated EBs are allowed to adhere to a laminin-coatedsurface, neuron-like outgrowths are observed and a result isdifferentiation into specific neuronal subtypes. Additional relevantdiscussion may be found in Davis-Dusenbery et al., 2014, How to makespinal motor neurons, Development 141(3):491-501; Sandoe and Eggan,2013, Opportunities and challenges of pluripotent stem cellneurodegenerative disease models, Nat Neuroscience 16(7):780-9; and Hanet al., 2011, Constructing and deconstructing stem cell models ofneurological disease, Neuron 70(4):626-44.

2b. Direct Conversion of Cells in Specific Cell Types

By pathway 211, human somatic cells are obtained and direct lineageconversion of the somatic cells into motor neurons may be performed.Conversion may include the use of lineage-specific transcription factorsto induce the conversion of specific cell types from unrelated somaticcells. See, e.g., Davis-Dusenbery et al., 2014, How to make spinal motorneurons, Development 141:491; Graf, 2011, Historical origins oftransdifferentiation and reprogramming, Cell Stem Cell 9:504-516. It hasbeen shown that a set of neural lineage-specific transcription factors,or BAM factors, causes the conversion of fibroblasts into inducedneuronal (iN) cells. Vierbuchen 2010 Nature 463:1035. MicroRNAs andadditional pro-neuronal factors, including NeuroD1, may cooperate withor replace the BAM factors during conversion of human fibroblasts intoneurons. See, for example, Ambasudhan et al., 2011, Direct reprogrammingof adult human fibroblasts to functional neurons under definedconditions, Cell Stem Cell 9:113-118; Pang et al., 2011, Induction ofhuman neuronal cells by defined transcription factors, Nature476:220-223; also see Yoo et al., 2011, MicroRNA mediated conversion ofhuman fibroblasts to neurons, Nature 476:228-231.

2c. Maintenance of Differentiated Cells

Differentiated cells such as motor neurons may be dissociated and platedonto glass coverslips coated with poly-d-lysine and laminin. Motorneurons may be fed with a suitable medium such as a neurobasal mediumsupplemented with N2, B27, GDNF, BDNF, and CTNF. Cells may be maintainedin a suitable medium such as an N2 medium (DMEM/F12 [1:1] supplementedwith laminin [1 μg/mL; Invitrogen], FGF-2 [10 ng/ml; R&D Systems,Minneapolis, Minn.], and N2 supplement [1%; Invitrogen]), furthersupplemented with GDNF, BDNF, and CNTF, all at 10 ng/ml. Suitable mediaare described in Son et al., 2011, Conversion of mouse and humanfibroblasts into functional spinal motor neurons, Cell Stem Cell9:205-218; Vierbuchen et al., 2010, Direct conversion of fibroblasts tofunctional neurons by defined factors, Nature4 63:1035-1041; Kuo et al.,2003, Differentiation of monkey embryonic stem cells into neurallineages, Biology of Reproduction 68:1727-1735; and Wernig et al., 2002,Tau EGFP embryonic stem cells: an efficient tool for neuronal lineageselection and transplantation. J Neuroscience Res 69:918-24, eachincorporated by reference.

3. Control Cell Line or Signature

Methods of the invention include causing the cell to express an opticalreporter, observing a signature generated by the optical reporter, andcomparing the observed signature to a control signature. The controlsignature may be obtained by obtaining a control cell that is also ofthe specific neural subtype and is genetically and phenotypicallysimilar to the test cells. In certain embodiments—where, for example, apatient has a known mutation or allele at a certain locus—geneticediting is performed to generate a control cell line that but for theknown mutation is isogenic with the test cell line. For example, where apatient is known to have the SOD1A4V mutation, genetic editingtechniques can introduce a SOD1V4A mutation into the cell line to createa control cell line with a wild-type genotype and phenotype. Genetic orgenome editing techniques may proceed via zinc-finger domain methods,transcription activator-like effector nucleases (TALENs), or clusteredregularly interspaced short palindromic repeat (CRISPR) nucleases.

Genome editing techniques (e.g., use of zinc finger domains) may be usedto create a control cell that is isogenic but-for a variant of interest.In certain embodiments, genome editing techniques are applied to the iPScells. For example, a second corrected line (SOD1V4A) may be generatedusing zinc finger domains resulting in two otherwise isogenic lines.After that, diseased and corrected iPS cells may be differentiated intomotor neurons using embryoid bodies according to the methods describedabove.

Genomic editing may be performed by any suitable method known in theart. For example, the chromosomal sequence encoding the target gene ofinterest may be edited using TALENs technology. TALENS are artificialrestriction enzymes generated by fusing a TAL effector DNA bindingdomain to a DNA cleavage domain. In some embodiments, genome editing isperformed using CRISPR technology. TALENs and CRISPR methods provideone-to-one relationship to the target sites, i.e. one unit of the tandemrepeat in the TALE domain recognizes one nucleotide in the target site,and the crRNA or gRNA of CRISPR/Cas system hybridizes to thecomplementary sequence in the DNA target. Methods can include using apair of TALENs or a Cas9 protein with one gRNA to generate double-strandbreaks in the target. The breaks are then repaired via non-homologousend-joining or homologous recombination (HR).

TALENs uses a nonspecific DNA-cleaving nuclease fused to a DNA-bindingdomain that can be to target essentially any sequence. For TALENtechnology, target sites are identified and expression vectors are made.See Liu et al, 2012, Efficient and specific modifications of theDrosophila genome by means of an easy TALEN strategy, J. Genet. Genomics39:209-215. The linearized expression vectors (e.g., by Notl) and usedas template for mRNA synthesis. A commercially available kit may be usesuch as the mMESSAGE mMACHINE SP6 transcription kit from LifeTechnologies (Carlsbad, Calif.). See Joung & Sander, 2013, TALENs: awideliy applicable technology for targeted genome editing, Nat Rev MolCell Bio 14:49-55.

CRISPR methodologies employ a nuclease, CRISPR-associated (Cas9), thatcomplexes with small RNAs as guides (gRNAs) to cleave DNA in asequence-specific manner upstream of the protospacer adjacent motif(PAM) in any genomic location. CRISPR may use separate guide RNAs knownas the crRNA and tracrRNA. These two separate RNAs have been combinedinto a single RNA to enable site-specific mammalian genome cuttingthrough the design of a short guide RNA. Cas9 and guide RNA (gRNA) maybe synthesized by known methods. Cas9/guide-RNA (gRNA) uses anon-specific DNA cleavage protein Cas9, and an RNA oligo to hybridize totarget and recruit the Cas9/gRNA complex. See Chang et al., 2013, Genomeediting with RNA-guided Cas9 nuclease in zebrafish embryos, Cell Res23:465-472; Hwang et al., 2013, Efficient genome editing in zebrafishusing a CRISPR-Cas system, Nat. Biotechnol 31:227-229; Xiao et al.,2013, Chromosomal deletions and inversions mediated by TALENS andCRISPR/Cas in zebrafish, Nucl Acids Res 1-11.

In certain embodiments, genome editing is performed using zinc fingernuclease-mediated process as described, for example, in U.S. Pub.2011/0023144 to Weinstein.

FIG. 3 gives an overview of a method 301 for zinc-finger nucleasemediated editing. Briefly, the method includes introducing into the iPScell at least one RNA molecule encoding a targeted zinc finger nuclease305 and, optionally, at least one accessory polynucleotide. The cellincludes target sequence 311. The cell is incubated to allow expressionof the zinc finger nuclease 305, wherein a double-stranded break 317 isintroduced into the targeted chromosomal sequence 311 by the zinc fingernuclease 305. In some embodiments, a donor polynucleotide or exchangepolynucleotide 321 is introduced. Target DNA 311 along with exchangepolynucleotide 321 may be repaired by an error-prone non-homologousend-joining DNA repair process or a homology-directed DNA repairprocess. This may be used to produce a control line with a controlgenome 315 that is isogenic to original genome 311 but for a changedsite. The genomic editing may be used to establish a control line (e.g.,where the patient is known to have a certain mutation, the zinc fingerprocess may revert the genomic DNA to wild type) or to introduce amutation (e.g., non-sense, missense, or frameshift) or to affecttranscription or expression.

Typically, a zinc finger nuclease comprises a DNA binding domain (i.e.,zinc finger) and a cleavage domain (i.e., nuclease) and this gene may beintroduced as mRNA (e.g., 5′ capped, polyadenylated, or both). Zincfinger binding domains may be engineered to recognize and bind to anynucleic acid sequence of choice. See, for example, Beerli & Barbas,2002, Engineering polydactyl zinc-finger transcription factors, Nat.Biotechnol, 20:135-141; Pabo et al., 2001, Design and selection of novelCys2His2 zinc finger proteins, Ann. Rev. Biochem 70:313-340; Isalan etal., 2001, A rapid generally applicable method to engineer zinc fingersillustrated by targeting the HIV-1 promoter, Nat. Biotechnol 19:656-660;and Santiago et al., 2008, Targeted gene knockout in mammalian cells byusing engineered zinc-finger nucleases, PNAS 105:5809-5814. Anengineered zinc finger binding domain may have a novel bindingspecificity compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. A zinc finger binding domain may be designedto recognize a target DNA sequence via zinc finger recognition regions(i.e., zinc fingers). See for example, U.S. Pat. Nos. 6,607,882;6,534,261 and 6,453,242, incorporated by reference. Exemplary methods ofselecting a zinc finger recognition region may include phage display andtwo-hybrid systems, and are disclosed in U.S. Pat. Nos. 5,789,538;5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; andU.S. Pat. No. 6,242,568, each of which is incorporated by reference.

Zinc finger binding domains and methods for design and construction offusion proteins (and polynucleotides encoding same) are known to thoseof skill in the art and are described in detail in U.S. Pub.2005/0064474 and U.S. Pub. 2006/0188987, each incorporated by reference.Zinc finger recognition regions, multi-fingered zinc finger proteins, orcombinations thereof may be linked together using suitable linkersequences, including for example, linkers of five or more amino acids inlength. See, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949,incorporated by reference.

The zinc finger nuclease may use a nuclear localization sequence (NLS).A NLS is an amino acid sequence which facilitates targeting the zincfinger nuclease protein into the nucleus to introduce a double strandedbreak at the target sequence in the chromosome. Nuclear localizationsignals are known in the art. See, for example, Makkerh, 1996,Comparative mutagenesis of nuclear localization signals reveals theimportance of neutral and acidic amino acids, Current Biology6:1025-1027.

A zinc finger nuclease also includes a cleavage domain. The cleavagedomain portion of the zinc finger nucleases may be obtained from anysuitable endonuclease or exonuclease such as restriction endonucleasesand homing endonucleases. See, for example, Belfort & Roberts, 1997,Homing endonucleases: keeping the house in order, Nucleic Acids Res25(17):3379-3388. A cleavage domain may be derived from an enzyme thatrequires dimerization for cleavage activity. Two zinc finger nucleasesmay be required for cleavage, as each nuclease comprises a monomer ofthe active enzyme dimer. Alternatively, a single zinc finger nucleasemay comprise both monomers to create an active enzyme dimer. Restrictionendonucleases present may be capable of sequence-specific binding andcleavage of DNA at or near the site of binding. Certain restrictionenzymes (e.g., Type IIS) cleave DNA at sites removed from therecognition site and have separable binding and cleavage domains. Forexample, the Type IIS enzyme FokI, active as a dimer, catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. The FokI enzyme used in a zinc finger nuclease may be considereda cleavage monomer. Thus, for targeted double-stranded cleavage using aFokI cleavage domain, two zinc finger nucleases, each comprising a FokIcleavage monomer, may be used to reconstitute an active enzyme dimer.See Wah, et al., 1998, Structure of FokI has implications for DNAcleavage, PNAS 95:10564-10569; U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994, each incorporated by reference. In certain embodiments, thecleavage domain may comprise one or more engineered cleavage monomersthat minimize or prevent homo-dimerization, as described, for example,in U.S. Patent Publication Nos. 2005/0064474, 2006/0188987, and2008/0131962, each incorporated by reference.

Genomic editing by the zinc finger nuclease-mediated process may includeintroducing at least one donor polynucleotide comprising a sequence intothe cell. A donor polynucleotide preferably includes the sequence to beintroduced flanked by an upstream and downstream sequence that sharesequence similarity with either side of the site of integration in thechromosome. The upstream and downstream sequences in the donorpolynucleotide are selected to promote recombination between thechromosomal sequence of interest and the donor polynucleotide.Typically, the donor polynucleotide will be DNA. The donorpolynucleotide may be a DNA plasmid, a bacterial artificial chromosome(BAC), a yeast artificial chromosome (YAC), a viral vector, a linearpiece of DNA, a PCR fragment, a naked nucleic acid, and may employ adelivery vehicle such as a liposome. The sequence of the donorpolynucleotide may include exons, introns, regulatory sequences, orcombinations thereof.

The double stranded break is repaired via homologous recombination withthe donor polynucleotide such that the desired sequence is integratedinto the chromosome.

In some embodiments, methods for genome editing include introducing intothe cell an exchange polynucleotide (typically DNA) with a sequence thatis substantially identical to the chromosomal sequence at the site ofcleavage and which further comprises at least one specific nucleotidechange. Where the cells have been obtained from a subject suspected tohave a neurodegenerative disease, a method such as TALENs, CRISPRs, orzinc fingers may be used to make a control cell line. For example, ifthe cell line is SOD1A4V, methods may be used to produce a cell linethat is isogenic but SOD1V4A. While any such technology may be used, thefollowing illustrates genome editing via zinc finger nucleases.

In general, with zinc-finger nucleases, the sequence of the exchangepolynucleotide will share enough sequence identity with the chromosomalsequence such that the two sequences may be exchanged by homologousrecombination. The sequence in the exchange polynucleotide comprises atleast one specific nucleotide change with respect to the sequence of thecorresponding chromosomal sequence. For example, one nucleotide in aspecific codon may be changed to another nucleotide such that the codoncodes for a different amino acid. In one embodiment, the sequence in theexchange polynucleotide may comprise one specific nucleotide change suchthat the encoded protein comprises one amino acid change.

In the zinc finger nuclease-mediated process for modifying a chromosomalsequence, a double stranded break introduced into the chromosomalsequence by the zinc finger nuclease is repaired, via homologousrecombination with the exchange polynucleotide, such that the sequencein the exchange polynucleotide may be exchanged with a portion of thechromosomal sequence. The presence of the double stranded breakfacilitates homologous recombination and repair of the break. Theexchange polynucleotide may be physically integrated or, alternatively,the exchange polynucleotide may be used as a template for repair of thebreak, resulting in the exchange of the sequence information in theexchange polynucleotide with the sequence information in that portion ofthe chromosomal sequence. Thus, a portion of the endogenous chromosomalsequence may be converted to the sequence of the exchangepolynucleotide.

To mediate zinc finger nuclease genomic editing, at least one nucleicacid molecule encoding a zinc finger nuclease and, optionally, at leastone exchange polynucleotide or at least one donor polynucleotide aredelivered to the cell of interest. Suitable methods of introducing thenucleic acids to the cell include microinjection, electroporation,calcium phosphate-mediated transfection, cationic transfection, liposometransfection, heat shock transfection, lipofection, and delivery vialiposomes, immunoliposomes, virosomes, or artificial virions.

The method of inducing genomic editing with a zinc finger nucleasefurther comprises culturing the cell comprising the introduced nucleicacid to allow expression of the zinc finger nuclease. Cells comprisingthe introduced nucleic acids may be cultured using standard proceduresto allow expression of the zinc finger nuclease. Typically, the cellsare cultured at an appropriate temperature and in appropriate media withthe necessary O2/CO2 ratio to allow the expression of the zinc fingernuclease. Suitable non-limiting examples of media include M2, M16, KSOM,BMOC, and HTF media. Standard cell culture techniques are described, forexample, in Santiago et al, 2008, Targeted gene knockout in mammaliancells by using engineered zinc finger nucleases, PNAS 105:5809-5814;Moehle et al., 2007, Targeted gene addition into a specified location inthe human genome using designed zinc finger nucleases PNAS104:3055-3060; Urnov et al., 2005, Highly efficient endogenous humangene correction using designed zinc-finger nucleases, Nature435(7042):646-51; and Lombardo et al., 2007, Gene editing in human stemcells using zinc finger nucleases and integrase-defective lentiviralvector delivery, Nat Biotechnol 25(11):1298-306. Those of skill in theart appreciate that methods for culturing cells are known in the art andcan and will vary depending on conditions. Upon expression of the zincfinger nuclease, the target sequence is edited. In cases in which thecell includes an expressed zinc finger nuclease as well as a donor (orexchange) polynucleotide, the zinc finger nuclease recognizes, binds,and cleaves the target sequence in the chromosome. The double-strandedbreak introduced by the zinc finger nuclease is repaired, via homologousrecombination with the donor (or exchange) polynucleotide, such that thesequence in the donor polynucleotide is integrated into the chromosomalsequence (or a portion of the chromosomal sequence is converted to thesequence in the exchange polynucleotide). As a consequence, a sequencemay be integrated into the chromosomal sequence (or a portion of thechromosomal sequence may be modified).

Using genome editing for modifying a chromosomal sequence, an isogenic(but for the mutation of interest) control line can be generated. Incertain embodiments, a control cells are obtained from healthyindividuals, i.e., without using genome editing on cells taken from thesubject. The control line can be used in the analytical methodsdescribed herein to generate a control signature for comparison to testdata. In some embodiments, a control signature is stored on-file afterhaving been previously generated and stored and the stored controlsignature is used (e.g., a digital file such as a graph or series ofmeasurements stored in a non-transitory memory in a computer system).For example, a control signature could be generated by assaying a largepopulation of subjects of known phenotype or genotype and storing anaggregate result as a control signature for later downstreamcomparisons.

4. Causing Cells to Express Optogenetic Systems

4a. Causing a Cell to Express an Optogenetic Reporter

The patient's test cell line and the optional control line may be causedto express an optical reporter of neural or electrical activity.Examples of neural activity include action potentials in a neuron orfusion of vesicles releasing neurotransmitters. Exemplary electricalactivity includes action potentials in a neuron, cardiomyocyte,astrocyte or other electrically active cell. Further examples of neuralor electrical activity include ion pumping or release or changing ionicgradients across membranes. Causing a cell to express an opticalreporter of neural activity can be done with a fluorescent reporter ofvesicle fusion. Expressing an optical reporter of neural or electricalactivity can include transformation with an optogenetic reporter. Forexample, the cell may be transformed with a vector comprising anoptogenetic reporter and the cell may also be caused to express anoptogenetic actuator by transformation. In certain embodiments, thedifferentiated neurons are cultured (e.g., for about 4 days) and theninfected with lentivirus bearing a genetically encoded optical reporterof neural activity and optionally an optical voltage actuator.

Any suitable optical reporter of neural activity may be used. Exemplaryreporters include fluorescent reporters of transmembrane voltagedifferences, pHluorin-based reporters of synaptic vesicle fusion, andgenetically encoded calcium indicators. In a preferred embodiment, agenetically encoded voltage indicator is used. Genetically encodedvoltage indicators that may be used or modified for use with methods ofthe invention include FlaSh (Siegel, 1997, A genetically encoded opticalprobe of membrane voltage. Neuron 19:735-741); SPARC (Ataka, 2002, Agenetically targetable fluorescent probe of channel gating with rapidkinetics, Biophys J 82:509-516); and VSFP1 (Sakai et al., 2001, Designand characterization of a DNA encoded, voltage-sensitive fluorescentprotein, Euro J Neuroscience 13:2314-2318). A genetically encodedvoltage indicator based on the paddle domain of a voltage-gatedphosphatase is CiVSP (Murata et al., 2005, Phosphoinositide phosphataseactivity coupled to an intrinsic voltage sensor, Nature 435:1239-1243).Another indicator is the hybrid hVOS indicator (Chanda et al., 2005, Ahybrid approach to measuring electrical activity in geneticallyspecified neurons, Nat Neuroscience 8:1619-1626), which transduces thevoltage dependent migration of dipicrylamine (DPA) through the membraneleaflet to “dark FRET” (fluorescence resonance energy transfer) with amembrane-targeted GFP.

Optical reporters that may be suitable for use with the inventioninclude those from the family of proteins of known microbial rhodopsins.A reporter based on a microbial rhodopsin may provide high sensitivityand speed. Suitable indicators include those that use the endogenousfluorescence of the microbial rhodopsin protein Archaerhodopsin 3 (Arch)from Halorubum sodomense. Arch resolves action potentials with highsignal-to-noise (SNR) and low phototoxicity. A mutant form of Arch,D95N, has been shown not to exhibit a hyperpolarizing current associatedwith some indicators. Other mutant forms of Arch, termed QuasAr1 andQuasAr2, have been shown to exhibit improved brightness, sensitivity tovoltage, speed of response, and trafficking to the neuronal plasmamembrane. Arch and the above-mentioned variants target eukaryoticmembranes and can image single action potentials and subthresholddepolarization in cultured mammalian neurons. See Kralj et al, 2012,Optical recording of action potentials in mammalian neurons using amicrobial rhodopsin, Nat Methods 9:90-95. Thus Arch and variants of Archsuch as Arch(D95N) may provide good optical reporters of neural activityaccording to embodiments of the invention.

In some embodiments, an improved variant of Arch such as QuasAr1 orQuasAr2 is used. QuasAr1 comprises Arch with the mutations: P60S, T80S,D95H, D106H, and F161V. QuasAr2 comprises Arch with the mutations: P60S,T80S, D95Q, D106H, and F161V. Positions Asp95 and Asp106 of Arch (whichare structurally aligned with positions Asp85 and Asp96 ofbacteriorhodopsin, and have been reported to play key roles in protontranslocation during the photo cycle) are targets for modificationbecause they flank the Schiff base in the proton-transport chain and arelikely important in determining voltage sensitivity and speed. The othermutations improve the brightness of the protein. Starting with an Archgene, it may be beneficial to add endoplasmic reticulum (ER) exportmotifs and a trafficking sequence (TS) according to methods known in theart.

FIG. 4 presents a structural model of Quasar1 based on homologousprotein Arch-2 (PDB: 2E14, described in Enami et al, 2006, Crystalstructures of archaerhodopsin-1 and -2: Common structural motif inArchaeal light-driven proton pumps, J Mol Bio. 358:675-685). MutationsT80S and F161V are located in the periphery of the protein, while P60Sis close to the Schiff base of the retinal chromophore. Given theirlocation, T80S and F161V substitutions are unlikely to have a directimpact on the photo-physical properties of the protein, and are morelikely to have a role in improving the folding efficiency. In contrast,the close proximity of the P60S substitution to the Schiff base suggeststhat this mutation has a more direct influence on the photo-physicalproperties. The QuasAr indicators may exhibit improved voltagesensitivity, response kinetics, membrane trafficking and diminisheddependence of brightness on illumination intensity relative to Arch. Thefluorescence quantum yields of solubilized QuasAr1 and 2 may be 19- and10-fold enhanced, respectively, relative to the non-pumping voltageindicator Arch(D95N). QuasAr1 may be 15-fold brighter than wild-typeArch, and QuasAr2 may be 3.3-fold brighter. Neither mutant shows theoptical nonlinearity seen in the wild-type protein. Fluorescence ofArch, QuasAr1, and QuasAr2 increase nearly linearly with membranevoltage between −100 mV and +50 mV. Fluorescence recordings may beacquired on an epifluorescence microscope, described in Kralj et al.,2012, Optical recording of action potentials in mammalian neurons usinga microbial rhodopsin, Nat. Methods 9:90-95.

QuasAr1 and QuasAr2 each refer to a specific variant of Arch. Asdiscussed, archaerhodopsin 3 (Arch) functions as a fast and sensitivevoltage indicator. Improved versions of Arch include the QuasArs(‘quality superior to Arch’), described in Hochbaum et al., 2014.QuasAr1 differs from wild-type Arch by the mutations P60S, T80S, D95H,D106H and F161V. QuasAr2 differed from QuasAr1 by the mutation H95Q.QuasAr1 and QuasAr2 report action potentials (APs).

FIG. 21 gives a comparison of AP waveforms as measured by thegenetically encoded voltage indicator QuasAr2 and the voltage-sensitivedye, FluoVolt. Cells are sparsely transfected with the QuasAr2 constructand then treated with FluoVolt dye. QuasAr2 is excited by red laserlight at a wavelength of 635 nm with fluorescence detection centered at720 nm. FluoVolt is excited by 488 nm laser light with fluorescencedetection centered at 525 nm. The top panel shows the simultaneouslyrecorded AP waveforms from a cell expressing QuasAr2 (red line) andlabeled with FluoVolt (green line). The similarity of these tracesestablishes that QuasAr2 fluorescence accurately represents theunderlying AP waveform. The lower trace compares the FluoVolt APwaveform in the presence (FluoVolt+, QuasAr2+, green) and absence(FluoVolt+, QuasAr2-, cyan) of QuasAr2 expression. The similarity ofthese two traces establishes that expression of QuasAr2 does not perturbthe AP waveform.

FIG. 22 shows plots of the average waveforms from the traces in FIG. 21.

FIG. 23 presents phototoxicity and photobleaching measurement ofQuasAr2. Cells are imaged under continuous red laser illumination (˜50W/cm2) for 500 s. Expanded views of the fluorescence recording are shownin the lower panels.

FIG. 24 graphs the average AP waveform shapes for the beginning (blue)and end (green) of the trace in FIG. 23.

Arch and the above-mentioned variants target eukaryotic membranes andcan image single action potentials and subthreshold depolarization incultured mammalian neurons. See Kralj et al, 2012, Optical recording ofaction potentials in mammalian neurons using a microbial rhodopsin, NatMethods 9:90-95 and Hochbaum et al., All-optical electrophysiology inmammalian neurons using engineered microbial rhodopsins, Nature Methods,11, 825-833 (2014), both incorporated by reference. Thus Arch andvariants of Arch may provide good optical reporters of electricalactivity according to embodiments of the invention.

The invention provides optical reporters based on Archaerhodopsins thatfunction in mammalian cells, including human stem cell-derived neurons.These proteins indicate electrical dynamics with sub-millisecondtemporal resolution and sub-micron spatial resolution and may be used innon-contact, high-throughput, and high-content studies of electricaldynamics in cells and tissues using optical measurement of membranepotential. These reporters are broadly useful, particularly ineukaryotic, such as mammalian, including human cells.

The invention includes reporters based on Archaerhodopsin 3 (Arch 3) andits homologues. Arch 3 is Archaerhodopsin from H. sodomense and it isknown as a genetically-encoded reagent for high-performanceyellow/green-light neural silencing. Gene sequence at GenBank:GU045593.1 (synthetic construct Arch 3 gene, complete cds. SubmittedSep. 28, 2009). These proteins localize to the plasma membrane ineukaryotic cells and show voltage-dependent fluorescence.

Fluorescence recordings may be acquired on an epifluorescencemicroscope, described in Hochbaum et al., All-optical electrophysiologyin mammalian neurons using engineered microbial rhodopsins, NatureMethods, 11, 825-833 (2014), incorporated by reference.

Optical reporters of the invention show high sensitivity. In mammaliancells, Archaerhodopsin-based reporters show about 3-fold increase influorescence between −150 mV and +150 mV. The response is linear overmost of this range. Membrane voltage can be measured with a precision of<1 mV in a 1 s interval. Reporters of the invention show high speed.QuasAr1 shows 90% of its step response in 0.05 ms. The upstroke of acardiac AP lasts approximately 1 ms, so the speeds of Arch-derivedindicators meet the benchmark for imaging electrical activity. Reportersof the invention show high photo-stability and are comparable to GFP inthe number of fluorescence photons produced prior to photobleaching. Thereporters may also show far red spectrum. The Arch-derivedvoltage-indicating protein reporters, sometimes referred to asgenetically encoded voltage indicators (GEVIs), may be excited with alaser at wavelengths between 590-640 nm, and the emission is in the nearinfrared, peaked at 710 nm. The emission is farther to the red than anyother existing fluorescent protein. These wavelengths coincide with lowcellular auto-fluorescence. This feature makes these proteinsparticularly useful in optical measurements of action potentials as thespectrum facilitates imaging with high signal-to-noise ratio, as well asmulti-spectral imaging in combination with other fluorescent probes.

Other optogenetic reporters may be used with methods and systems of theinvention. Suitable optogenetic reporters include the two Arch variantsdubbed Archer1 and Archer2 reported in Flytzanis, et al., 2014,Archaerohodopsin variants with enhanced voltage-sensitive fluorescencein mammalian and Caenorhabditis elegans neurons, Nat Comm 5:4894,incorporated by reference. Archer1 and Archer2 exhibit enhanced radiancein response to 655 nm light have 3-5 times increased fluorescence and55-99 times reduced photocurrents compared with Arch WT. Archer1 (D95Eand T99C) and Archer2 (D95E, T99C and A225M) may be used for voltagesensing. These mutants exhibit high baseline fluorescence (×3-5 overArch WT), large dynamic range of sensitivity (85% DF/F and 60% DF/F per100 mV for Archer1 and Archer2, respectively) that is stable over longillumination times, and fast kinetics, when imaged at ×9 lower lightintensity (880 mW mm^−2 at 655 nm) than the most recently reported Archvariants. Archer1's characteristics allow its use to monitor rapidchanges in membrane voltage throughout a single neuron and throughout apopulation of neurons in vitro. Although Archer1 has minimal pumping atwavelengths used for fluorescence excitation (655 nm), it maintainsstrong proton pumping currents at lower wavelengths (560 nm). Archer1provides a bi-functional tool with both voltage sensing with red lightand inhibitory capabilities with greenlight. Archer1 is capable ofdetecting small voltage changes in response to sensory stimulus

Suitable optogenetic reporters include the Arch-derived voltage sensorswith trafficking signals for enhanced localization as well as the Archmutants dubbed Arch-EEN and Arch-EEQ reported in Gong et al., EnhancedArchaerhodopsin fluorescent protein voltage indicators, PLoSOne8(6):e66959, incorporated by reference. Such reporters may includevariants of Arch with the double mutations D95N-D106E (Arch-EEN) andD95Q-D106E (Arch-EEQ).

Suitable optogenetic reporters include sensors that use fluorescenceresonance energy transfer (FRET) to combine rapid kinetics and thevoltage dependence of the rhodopsin family voltage-sensing domains withthe brightness of genetically engineered protein fluorophores. SuchFRET-opsin sensors offer good spike detection fidelity, fast kinetics,and high brightness. FRET-opsin sensors are described in Gong et al.,Imaging neural spiking in brain tissue using FRET-opsin protein voltagesensors, Nat Comm 5:3674, incorporated by reference. A suitableFRET-opsin may include a fusion of a bright fluorophore to act as a FRETdonor to a Mac rhodopsin molecule to server as both the voltage sensingdomain and the FRET acceptor. Other sensors include the AcceleratedSensor of Action Potentials (ASAP1), a voltage sensor formed byinsertion of a circularly permuted GFP into a chicken voltage-sensitivephosphatase. St-Pierre, 2014, High-fidelity optical reporting ofneuronal electrical activity with an ultrafast fluorescent voltagesensor, Nat Neurosci 17(6):884, incorporated by reference. Othersuitable reporters may include the ArcLight-derived probe dubbedBongwoori and described in Piao et al., 2015, Combinatorial mutagenesisof the voltage-sensing domain enables the optical resolution of actionpotentials firing at 60 Hz by a genetically encoded fluorescent sensorof membrane potential, J Neurosci 35(1):372-385, incorporated byreference.

4b. Causing a Cell to Express an Optogenetic Actuator

In a preferred embodiment, the cells are transformed with an opticalvoltage actuator. This can occur, for example, simultaneously withtransformation with the vector comprising the optogenetic reporter. Thefar-red excitation spectrum of the QuasAr reporters suggests that theymay be paired with a blue light-activated channelrhodopsin to achieveall-optical electrophysiology. For spatially precise optical excitation,the channelrhodopsin should carry current densities sufficient to induceAPs when only a subsection of a cell is excited. Preferably, light usedfor imaging the reporter should not activate the actuator, and lightused for activating the actuator should not confound the fluorescencesignal of the reporter. Thus in a preferred embodiment, an opticalactuator and an optical reporter are spectrally orthogonal to avoidcrosstalk and allow for simultaneous use. Spectrally orthogonal systemsare discussed in Carlson and Campbell, 2013, Circular permutated redfluorescent proteins and calcium ion indicators based on mCherry,Protein Eng Des Sel 26(12):763-772.

Preferably, a genetically-encoded optogenetic actuator is used. Oneactuator is channelrhodopsin2 H134R, an optogenetic actuator describedin Nagel, G. et al., 2005, Light activation of channelrhodopsin-2 inexcitable cells of Caenorhabditis elegans triggers rapid behavioralresponses, Curr. Biol. 15, 2279-2284.

A screen of plant genomes has identified an optogenetic actuator,Scherffelia dubia ChR (sdChR), derived from a fresh-water green algafirst isolated from a small pond in Essex, England. See Klapoetke etal., 2014, Independent optical excitation of distinct neuralpopulations, Nat Meth Advance Online Publication 1-14; see alsoMelkonian & Preisig, 1986, A light and electron microscopic study ofScherffelia dubia, a new member of the scaly green flagellates(Prasinophyceae). Nord. J. Bot. 6:235-256, both incorporated byreference. SdChR may offer good sensitivity and a blue action spectrum.

An improved version of sdChR dubbed CheRiff may be used as an opticalactuator. The gene for Scherffelia dubia Channelrhodopsin (sdChR)(selected from a screen of channelrhodopsins for its blue excitationpeak (474 nm) and its large photocurrent relative to ChR2) issynthesized with mouse codon optimization, a trafficking sequence fromKir2.1 is added to improve trafficking, and the mutation E154A isintroduced. CheRiff exhibits significantly decreased crosstalk from redillumination (to 10.5±2.8 pA) allowing its use in cells along withoptogenetic reporters described herein. CheRiff shows good expressionand membrane trafficking in cultured rat hippocampal neurons. Themaximum photocurrent under saturating illumination (488 nm, 500 mW/cm)is 2.0±0.1 nA (n=10 cells), approximately 2-fold larger than the peakphotocurrents of ChR2 H134R or ChIEF (Lin et al., 2009, Characterizationof engineered channelrhodopsin variants with improved properties andkinetics, Biophys J 96:1803-1814). In neurons expressing CheRiff,whole-cell illumination at only 22±10 mW/cm induces a photocurrent of 1nA. Compared to ChR2 H134R and to ChIEF under standard channelrhodopsinillumination conditions (488 nm, 500 mW/cm). At 23° C., CheRiff reachespeak photocurrent in 4.5±0.3 ms (n=10 cells). After a 5 ms illuminationpulse, the channel closing time constant was comparable between CheRiffand ChIEF (16±0.8 ms, n=9 cells, and 15±2 ms, n=6 cells, respectively,p=0.94), and faster than ChR2 H134R (25±4 ms, n=6 cells, p<0.05). Undercontinuous illumination CheRiff partially desensitizes with a timeconstant of 400 ms, reaching a steady-state current of 1.3±0.08 nA (n=10cells). Illumination of neurons expressing CheRiff induces trains of APswith high reliability and high repetition-rate.

When testing for optical crosstalk between QuasArs and CheRiff incultured neurons, illumination sufficient to induce high-frequencytrains of APs (488 nm, 140 mW/cm) perturbed fluorescence of QuasArs by<1%. Illumination with high intensity red light (640 nm, 900 W/cm)induced an inward photocurrent through CheRiff of 14.3±3.1 pA, whichdepolarized neurons by 3.1±0.2 mV (n=5 cells). ChIEF and ChR2 H134Rgenerated similar red light photocurrents and depolarizations. For mostapplications this level of optical crosstalk is acceptable.

In some embodiments it is preferred to have an actuator whose activationis maximal at a violet light wavelength between 400-440 nm, further tothe blue than CheRiff. Violet-activated channelrhodopsins can besimultaneously combined with yellow-excited Ca2+ indicators (e.g.jRCaMP1a, jRGECO1a, and R-CaMP2) and a red-excited voltage indicator,e.g. QuasAr2, for simultaneous monitoring of Ca2+ and voltage underoptical stimulus conditions.

A preferred violet-excited channelrhodopsin actuator is TsChR, derivedfrom Tetraselmis striata (See Klapoetke et al., 2014, Independentoptical excitation of distinct neural populations, Nat. Meth. 11,338-346 (2014)). This channelrhodopsin actuator has a blue-shiftedaction spectrum with a peak at 435 nm. Another preferred violetchannelrhodopsin actuator is PsChR, derived from Platymonassubcordiformis (see Govorunova, Elena et al., 2013, Characterization ofa highly efficient blue-shifted channelrhodopsin from the marine algaPlatymonas subcordiformis, J Biol Chem 288(41):29911-29922). PsChr has ablue-shifted action spectrum with a peak at 437 nm. PsChR and TsChR areadvantageously paired with red-shifted Ca2+ indicators and can be usedin the same cell or same field of view as these red-shifted Ca2+indicators without optical crosstalk.

4c. Vectors for Expression of Optogenetic Systems

The optogenetic reporters and actuators may be delivered in constructsdescribed here as optopatch constructs delivered through the use of anexpression vector. Optopatch may be taken to refer to systems thatperform functions traditionally associated with patch clamps, but via anoptical input, readout, or both as provided for by, for example, anoptical reporter or actuator. An Optopatch construct may include abicistronic vector for co-expression of CheRiff-eGFP and QuasAr1- orQuasAr2-mOrange2. The QuasAr and CheRiff constructs may be deliveredseparately, or a bicistronic expression vector may be used to obtain auniform ratio of actuator to reporter expression levels.

The genetically encoded reporter, actuator, or both may be delivered byany suitable expression vector using methods known in the art. Anexpression vector is a specialized vector that contains the necessaryregulatory regions needed for expression of a gene of interest in a hostcell. In some embodiments the gene of interest is operably linked toanother sequence in the vector. In some embodiments, it is preferredthat the viral vectors are replication defective, which can be achievedfor example by removing all viral nucleic acids that encode forreplication. A replication defective viral vector will still retain itsinfective properties and enters the cells in a similar manner as areplicating vector, however once admitted to the cell a replicationdefective viral vector does not reproduce or multiply. The term“operably linked” means that the regulatory sequences necessary forexpression of the coding sequence are placed in the DNA molecule in theappropriate positions relative to the coding sequence so as to effectexpression of the coding sequence. This same definition is sometimesapplied to the arrangement of coding sequences and transcription controlelements (e.g. promoters, enhancers, and termination elements) in anexpression vector.

Many viral vectors or virus-associated vectors are known in the art.Such vectors can be used as carriers of a nucleic acid construct intothe cell. Constructs may be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others,including retroviral and lentiviral vectors, for infection ortransduction into cells. The vector may or may not be incorporated intothe cell's genome. The constructs may include viral sequences fortransfection, if desired. Alternatively, the construct may beincorporated into vectors capable of episomal replication, such as anEptsein Barr virus (EPV or EBV) vector. The inserted material of thevectors described herein may be operatively linked to an expressioncontrol sequence when the expression control sequence controls andregulates the transcription and translation of that polynucleotidesequence. In some examples, transcription of an inserted material isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene. In some embodiments, a recombinant cell containing an induciblepromoter is used and exposed to a regulatory agent or stimulus byexternally applying the agent or stimulus to the cell or organism byexposure to the appropriate environmental condition or the operativepathogen. Inducible promoters initiate transcription only in thepresence of a regulatory agent or stimulus. Examples of induciblepromoters include the tetracycline response element and promotersderived from the beta-interferon gene, heat shock gene, metallothioneingene or any obtainable from steroid hormone-responsive genes. Induciblepromoters which may be used in performing the methods of the presentinvention include those regulated by hormones and hormone analogs suchas progesterone, ecdysone and glucocorticoids as well as promoters whichare regulated by tetracycline, heat shock, heavy metal ions, interferon,and lactose operon activating compounds. See Gingrich and Roder, 1998,Inducible gene expression in the nervous system of transgenic mice, AnnuRev Neurosci 21:377-405. Tissue specific expression has been wellcharacterized in the field of gene expression and tissue specific andinducible promoters are well known in the art. These promoters are usedto regulate the expression of the foreign gene after it has beenintroduced into the target cell. In certain embodiments, a cell-typespecific promoter or a tissue-specific promoter is used. A cell-typespecific promoter may include a leaky cell-type specific promoter, whichregulates expression of a selected nucleic acid primarily in one celltype, but cause expression in other cells as well. For expression of anexogenous gene specifically in neuronal cells, a neuron-specific enolasepromoter can be used. See Forss-Petter et al., 1990, Transgenic miceexpressing beta-galactosidase in mature neurons under neuron specificenolase promoter control, Neuron 5: 187-197. For expression of anexogenous gene in dopaminergic neurons, a tyrosine hydroxylase promotercan be used.

In some embodiments, the expression vector is a lentiviral vector.Lentiviral vectors may include a eukaryotic promoter. The promoter canbe any inducible promoter, including synthetic promoters, that canfunction as a promoter in a eukaryotic cell. For example, the eukaryoticpromoter can be, but is not limited to, CamKIIα promoter, human Synapsinpromoter, ecdysone inducible promoters, Ela inducible promoters,tetracycline inducible promoters etc., as are well known in the art. Inaddition, the lentiviral vectors used herein can further comprise aselectable marker, which can comprise a promoter and a coding sequencefor a selectable trait. Nucleotide sequences encoding selectable markersare well known in the art, and include those that encode gene productsconferring resistance to antibiotics or anti-metabolites, or that supplyan auxotrophic requirement. Examples of such sequences include, but arenot limited to, those that encode thymidine kinase activity, orresistance to methotrexate, ampicillin, kanamycin, among others. Use oflentiviral vectors is discussed in Wardill et al., 2013, A neuron-basedscreening platform for optimizing genetically-encoded calciumindicators, PLoS One 8(10):e77728; Dottori, et al., Neural developmentin human embryonic stem cells-applications of lentiviral vectors, J CellBiochem 112(8):1955-62; and Diester et al., 2011, An optogenetic toolboxdesigned for primates, Nat Neurosci 14(3):387-97. When expressed under aCaMKIIα promoter in cultured rat hippocampal neurons the Optopatchconstruct exhibits high expression and good membrane trafficking of bothCheRiff and QuasAr2.

In some embodiments the viral vector is an adeno-associated virus (AAV)vector. AAV can infect both dividing and non-dividing cells and mayincorporate its genome into that of the host cell. One suitable viralvector uses recombinant adeno-associated virus (rAAV), which is widelyused for gene delivery in the CNS.

In certain embodiments, methods of the invention use a Cre-dependentexpression system. Cre-dependent expression includes Cre-Loxrecombination, a site-specific recombinase technology that uses theenzyme Cre recombinase, which recombines a pair of short targetsequences called the Lox sequences. This system can be implementedwithout inserting any extra supporting proteins or sequences. The Creenzyme and the original Lox site called the LoxP sequence are derivedfrom bacteriophage P1. Bacteriophage P1 uses Cre-lox recombination tocircularize and replicate its genomic DNA. This recombination strategyis employed in Cre-Lox technology for genome manipulation, whichrequires only the Cre recombinase and LoxP sites. Sauer & Henderson,1988, Site-specific DNA recombination in mammalian cells by the Crerecombinase of bacteriophage P1, PNAS 85:5166-70 and Sternberg &Hamilton, 1981, Bacteriophage P1 site-specific recombination. I.Recombination between LoxP sites, J Mol Biol 150:467-86. Methods may usea Cre recombinase-dependent viral vector for targeting tools such aschannelrhodopsin-2 (ChR2) to specific neurons with expression levelssufficient to permit reliable photostimulation. Optogenetic tools suchas ChR2 tagged with a fluorescent protein such as mCherry (e.g.,ChR2mCherry) or any other of the tools discussed herein are thusdelivered to the cell or cells for use in characterizing those cells.

The delivery vector may include Cre and Lox. The vector may furtheroptionally include a Lox-stop-Lox (LSL) cassette to prevent expressionof the transgene in the absence of Cre-mediated recombination. In thepresence of Cre recombinase, the LoxP sites recombine, and a removabletranscription termination Stop element is deleted. Removal of the stopelement may be achieved through the use of AdenoCre, which allowscontrol of the timing and location of expression. Use of the LSLcassette is discussed in Jackson, et al., 2001, Analysis of lung tumorinitiation and progression using conditional expression of oncogenicK-ras, Genes & Dev 15:3243-3248.

In certain embodiments, a construct of the invention uses a“flip-excision” switch, or FLEX switch (FLip EXicision), to achievestable transgene inversion. The FLEX switch is discussed in Schnutgen etal., 2003, A directional strategy for monitoring Cre-mediatedrecombination at the cellular level in the mouse, Nat Biotechnol21:562-565. The FLEX switch uses two pairs of heterotypic, antiparallelLoxP-type recombination sites which first undergo an inversion of thecoding sequence followed by excision of two sites, leading to one ofeach orthogonal recombination site oppositely oriented and incapable offurther recombination. A FLEX switch provides high efficiency andirreversibility. Thus in some embodiments, methods use a viral vectorcomprising rAAV-FLEX-rev-ChR2mCherry. Additionally or alternatively, avector may include FLEX and any other optogenetic tool discussed herein(e.g., rAAV-FLEX-QuasAr, rAAV-FLEX-CheRiff). UsingrAAV-FLEX-rev-ChR2mCherry as an illustrative example, Cre-mediatedinversion of the ChR2mCherry coding sequence results in the codingsequence being in the wrong orientation (i.e., rev-ChR2mCherry) fortranscription until Cre inverts the sequence, turning on transcriptionof ChR2mCherry. FLEX switch vectors are discussed in Atasoy et al.,2009, A FLEX switch targets channelrhodopsin-2 to multiple cell typesfor imaging and long-range circuit mapping, J Neurosci 28(28):7025-7030.

Use of a viral vector such as Cre-Lox system with an optical reporter,optical actuator, or both (optionally with a FLEX switch and/or aLox-Stop-Lox cassette) for labeling and stimulation of neurons allowsfor efficient photo-stimulation with only brief exposure (1 ms) to lessthan 100 μW focused laser light or to light from an optical fiber. SuchFurther discussion may be found in Yizhar et al., 2011, Optogenetics inneural systems, Neuron 71(1):9-34; Cardin et al., 2010, Targetedoptogenetic stimulation and recording of neurons in vivo usingcell-type-specific expression of Channelrhodopsin-2, Nat Protoc5(2):247-54; Rothermel et al., 2013, Transgene expression intarget-defined neuron populations mediated by retrograde infection ithadeno-associated viral vectors, J Neurosci 33(38):195-206; and Saunderset al., 2012, Novel recombinant adeno-associated viruses for Creactivated and inactivated transgene expression in neurons, Front NeuralCircuits 6:47.

In certain embodiments, actuators, reporters, or other genetic materialmay be delivered using chemically-modified mRNA. It may be found andexploited that certain nucleotide modifications interfere withinteractions between mRNA and toll-like receptor, retinoid-induciblegene, or both. Exposure to mRNAs coding for the desired product may leadto a desired level of expression of the product in the cells. See, e.g.,Kormann et al., 2011, Expression of therapeutic proteins after deliveryof chemically modified mRNA in mice, Nat Biotech 29(2):154-7; Zangi etal., 2013, Modified mRNA directs the fate of heart protenitor cells andinduces vascular regeneration after myocardial infarction, Nat Biotech31:898-907.

It may be beneficial to culture or mature the cells after transformationwith the genetically encoded optical reporter with optional actuator. Insome embodiments, the neurons are matured for 8-10 days post infection.Using microscopy and analytical methods described herein, the cell andits action potentials may be observed. For additional discussion, seeU.S. Pub. 2013/0224756, incorporated by reference in its entirety forall purposes.

4d. Optogenetic Constructs and Plating Schemes for Simultaneous Voltageand Ca2+ Measurement.

FIG. 25 presents schematic structures of optogenetic proteins used forstimulus and detection of voltage and intracellular Ca2+. The diagramsshow proteins homologous to CheRiff and QuasAr2. Stimulus of cells isachieved through 488 nm LED illumination of CheRiff. The CheRiffconstruct is coupled to an eGFP tag for detection of CheRiff expression.A fusion protein called CaViar (Hou et al., 2014), consisting of QuasAr2(Hochbaum et al., 2014) fused to GCaMP6f (Chen et al., 2013), is usedfor simultaneous voltage and Ca2+ imaging. QuasAr2 is excited via redlaser light. GCaMP6f is excited via blue laser light. Cells areseparately transduced with either CheRiff or CaViar vectors.

FIG. 26 illustrates cellular plating configurations. For simultaneousoptical stimulus and voltage imaging, CheRiff cells (solid cyan circles)are co-mingled with CaViar cells (solid red circles). The yellow dottedline indicates a microscope field of view. For simultaneous opticalstimulus and imaging of both Ca2+ and membrane voltage, cells are platedto spatially segregate CheRiff-expressing cells from CaViar-expressingcells to avoid optical crosstalk between the pulsed blue light used toperiodically stimulate the CheRiff-expressing cells and the continuousblue light used to image the CaViar-expressing cells. TheCheRiff-expressing cells lay outside the imaging region.

When testing for optical crosstalk between Arch-based reporters andCheRiff in cultured cells, illumination sufficient to induce APs (488nm, 140 mW/cm2) perturbed fluorescence of QuasAr reporters by <1%.Illumination with high intensity red light (640 nm, 900 W/cm2) inducedan inward photocurrent through CheRiff of 14.3±3.1 pA, which depolarizedcells by 3.1±0.2 mV (n=5 cells). ChIEF and ChR2 H134R generated similarred light photocurrents and depolarizations. For most applications thislevel of optical crosstalk is acceptable.

4e. Multimodal Sensing/Multiplexing

Membrane potential is only one of several mechanisms of signaling withincells. One may correlate changes in membrane potential with changes inconcentration of other species, such as Ca2+, H+ (i.e. pH), Na+, ATP,cAMP, NADH. We constructed fusions of Arch with pHluorin (a fluorescentpH indicator) and GCaMP6f (a fluorescent Ca2+ indicator). The fusion ofan Arch-based voltage indicator and a genetically encoded Ca2+ indicatoris called CaViar (See Hou et al., 2014, Simultaneous mapping of membranevoltage and calcium in zebrafish heart in vivo reveals chamber-specificdevelopmental transitions in ionic currents, Frontiers in physiology 5).One can also use fusions with other protein-based fluorescent indicatorsto enable other forms of multimodal imaging using the concept as taughtherein. Concentration of ions such as sodium, potassium, chloride, andcalcium can be simultaneously measured when the nucleic acid encodingthe microbial rhodopsin is operably linked to or fused with anadditional fluorescent analyte sensitive indicator; or when themicrobial rhodopsin and the additional fluorescent analyte sensitiveindicator are co-expressed in the same cell.

It is often desirable to achieve simultaneous optical stimulation of acell, calcium imaging, and voltage imaging. To achieve all threemodalities in the same cell, the invention provides for a violet-excitedChannelrhodopsin actuator (psChR or TsChR); a red-shifted geneticallyencoded calcium indicator; and a far red Arch-derived voltage indicator.Red-shifted genetically encoded calcium indicators include R-GECO1 (SeeZhao, Yongxin, et al. “An expanded palette of genetically encoded Ca2+indicators.” Science 333.6051 (2011): 1888-1891 and Wu, Jiahui, et al.“Improved orange and red Ca2+ indicators and photophysicalconsiderations for optogenetic applications.” ACS chemical neuroscience4.6 (2013): 963-972, both incorporated by reference), R-CaMP2 (SeeInoue, Masatoshi, et al. “Rational design of a high-affinity, fast, redcalcium indicator R-CaMP2.” Nature methods 12.1 (2015): 64-70,incorporated by reference), jRCaMP1a (Addgene plasmid 61562), andjRGECO1a (Addgene plasmid 61563). These calcium indicators are excitedby wavelengths between 540 and 560 nm, and emit at wavelengths between570 and 620 nm, thereby permitting spectral separation from theviolet-excited channelrhodopsin actuator and the Arch-based voltageindicator.

One can combine imaging of voltage indicating proteins with otherstructural and functional imaging, of e.g. pH, calcium, or ATP. One mayalso combine imaging of voltage indicating proteins with optogeneticcontrol of membrane potential using e.g. channelrhodopsin,halorhodopsin, and Archaerhodopsin. If optical measurement and controlare combined, one can perform all-optical electrophysiology to probe thedynamic electrical response of any membrane.

The invention provides high-throughput methods of characterizing cells.Robotics and custom software may be used for screening large librariesor large numbers of conditions which are typically encountered in highthroughput drug screening methods.

4f Optical Readout

Embodiments of the invention provide for spatial separation ofstimulating cells and reporter cells. Expression ofchannelrhodopsin-based light-gated ion channels provides a means toachieve optical stimulus. However, the blue light used to activate thesechannels may overlap spectrally with the light used to image mostsmall-molecule and genetically encoded fluorescent reporters ofphysiological activity (e.g. gCaMP Ca2+ indicators, Percival ATPindicators, pHluorin pH indicators, VF2.1.C1 voltage-sensitive dyes).Also, the light used to image these reporters may lead to off-targetactivation of all known channelrhodopsin actuators. Ideally, one wouldlike to optically stimulate a cell culture while maintaining freedom torecord from fluorescent reporters of any color, without opticalcrosstalk between the stimulus and the physiological measurement.Methods of the invention allow a cellular culture to be opticallystimulated while also using fluorescent reporters of any color, withoutoptical crosstalk between the stimulus and the physiological measurementthrough the spatial separation of actuator cells and reporter cells.

One solution presented here comprises expressing channelrhodopsinactuators in one set of hiPSC-derived cells, and expressing reporters(e.g. CaViar dual-function Ca2+ and voltage reporter) in another set ofcells. Flashes of blue light are delivered to the actuator cells, whilecontinuous blue light is used to monitor the reporter cells. Theactuator cells stimulate the reporter cells through synapses. A keychallenge is to identify and target the stimulus and the measurementlight beams to the appropriate corresponding cells. Methods of theinvention provide at least two embodiments of the solution to theproblem of targeting separate stimulus and measurement light beams tothe appropriate cells: a first approach based on spatial segregation anda second approach based on image processing and patterned illumination.

4g. Spatial Segregation

In a first embodiment using spatial segregation, light is targeted tothe actuator cells using spatial segregation of actuator andreporter-expressing cells.

Cells are independently infected with actuator and reporter and arere-plated in distinct but electrically contiguous regions. Opticalstimulus is delivered only to regions of the dish with cells expressingthe actuator, and sensor measurements using any wavelength of light arerecorded in regions of the dish away from cells expressing the actuator.In one instance, the actuator is CheRiff, and the sensor is CaViar inhuman iPSC-derived neurons.

FIG. 27 shows cells expressing CheRiff plated in an annular region, 10mm outer diameter, ˜8 mm diameter. The inner radius is set by a disk ofpolydimethyl siloxane (PDMS) adhered to the coverslip and the outerdiameter is set by the edge of the chamber. The PDMS disk is thenremoved and cells expressing CaViar are plated throughout. Stimulus iscontrolled by a blue LED whose illumination is confined to a smallregion of the actuating cells. Voltage and calcium imaging are achievedwith a red and blue laser, respectively, in a region free ofCheRiff-expressing cells.

4h. Patterned Illumination

In a second embodiment using patterned illumination, light is targetedto the actuator cells using image processing and patterned illuminationto separately target intermingled actuator- and reporter-expressingcells.

For image processing and patterned illumination, cells expressing eitheractuator or reporters are randomly intermixed. In one embodiment, cellsare initially plated separately and caused to express either theactuator or the reporter. The cells are then lifted from theirrespective dishes, mixed, and co-plated onto the imaging dish. Inanother embodiment, cells are plated directly in the imaging chamber,and doubly infected with lentivirus encoding Cre-On actuator and aCre-Off reporter. The cells are then infected sparsely with lentivirusencoding the Cre protein, so that in a sparse subset of cells theactuator is switched on and the reporter is switched off.

Cells expressing the actuator are identified via a recognizable marker,e.g. a fluorescent protein, or by their absence of fluorescencetransients indicating presence of a reporter. Optical stimulus isachieved by spatially patterning the excitation light using a digitalmicromirror device (DMD) to project flashes onto only those cellsexpressing the actuator.

FIG. 5 diagrams an optical imaging apparatus 501 for patternedillumination. A 488 nm blue laser beam is modulated in intensity by anacousto-optic modulator (not shown), and then reflected off a digitalmicromirror device (DMD) 505. The DMD imparted a spatial pattern on theblue laser beam (used for CheRiff excitation) on its way into themicroscope. The micromirrors are re-imaged onto the sample 509, leadingto an arbitrary user-defined spatio-temporal pattern of illumination atthe sample. Simultaneous whole-field illumination with 640 nm red lightexcites fluorescence of the reporter.

The fluorescent protein serving as a recognizable marker of the cellsexpressing the actuator is imaged to determine a pattern of thoseactuator cells. The digital coordinates of that image are used tocontrol the DMD 505 so that the DMD 505 directs the blue 488 nm lightonly onto the actuator cells. Due to the precision of the patternedillumination provided by the DMD 505, the cells expressing the reporterare not exposed to the 488 nm light. Cells expressing the reporter areimaged under continuous illumination, with the 640 nm light targeted viathe DMD to illuminate only those cells expressing the reporter, andoptionally continuous illumination at a wavelength of 488 nm toilluminate an additional reporter such as a GCaMP calcium indicator.

By the patterned illumination method, flashes of blue light aredelivered to the actuator cells, while continuous red and/or blue lightis used to monitor the reporter cells. The actuator cells stimulate thereporter cells (e.g., across synapses). Preferably, the actuator cellscomprise a first set of hiPSC-derived neurons expressingchannelrhodopsin actuators and the reporter cells comprise a second setof hiPSC-derived neurons expressing reporters (e.g. QuasAr2 or CaViardual-function Ca2+ and voltage reporter).

The foregoing (i) spatial segregation and (ii) patterned illuminationmethods provide for optical detection of changes in membrane potential,[Ca2+], or both, in optically stimulated neurons. The described methodsand techniques herein provide for the optical detection of the effectsof compounds on cells such as cells with disease genotypes. Suchdetection allows for evaluating the effect of a compound or otherstimulus on the phenotype of such cells.

4i. Preparation of Plates for Voltage Imaging

MatTek dishes (MatTek corp.; 10 mm glass diameter, #1.5) are coated with10 μg/mL fibronectin (Sigma-Aldrich) in 0.1% gelatin overnight at 4° C.Trypsinized CaViar and CheRiff-expressing cells are first mixed at aratio of 5:1 CaViar:CheRiff, and then pelleted. The combined cells arere-suspended in 2.1 mL of maintenance medium and plated at a density of2.5×104 cells/cm2 in 100 μL of plating medium to cover the entire glasssurface. Cells are kept at 37° C. in 5% CO2 overnight to adhere to theglass. Maintenance medium (1.0 mL) is added to each dish and the cellsare fed every 48 hours by removing 750 μL of medium from the dish andreplacing with 750 μL fresh maintenance medium.

4j. Preparation of Plates for Simultaneous Voltage and Calcium Imaging

For simultaneous voltage and calcium imaging, MatTek dishes (10 mm glassdiameter) are prepared to segregate CheRiff-expressing cells fromCaViar-expressing cells. This allows simultaneous calcium imaging andCheRiff stimulus, both with blue light, without optical crosstalkbetween the two functions. In certain embodiments, 8 mm-diameterpoly-dimethylsiloxane (PDMS) discs are treated with a solution of 10μg/mL fibronectin in 0.1% gelatin on one side for 10 minutes at roomtemperature. The coated discs are then dried and then pressed onto theMatTek dish glass surface, slightly offset to one side. The remainingexposed area of the glass is then coated with 10 μg/mL fibronectin in0.1% gelatin. Cells expressing the CheRiff are trypsinized according tothe manufacturer's protocol and re-suspended in 50 μL of maintenancemedium per dish. For plating, 50 μL of the CheRiff cells are then addedto the exposed portion of the glass surface and allowed to sit for 40minutes at 37° C. in 5% CO2 to allow the cells to adhere. The PDMS discsare then removed, the glass surface washed with 150 μL of maintenancemedia medium and the remaining volume aspirated. Trypsinized CaViarcells are then re-suspended in 100 μL of maintenance medium per dish andplated at a density of 2.0×104 cells/cm2 in 100 μL to cover the entireglass surface. Cells are kept at 37° C. in 5% CO2 overnight to adhere tothe glass. 1.0 0 mL of maintenance medium is added to each dish and thecells are fed every 48 hours by removing 750 μL of media from the dishand adding 750 μL fresh maintenance medium.

5. Imaging Activity Assay

5a. Capturing Images

Methods of the invention may include stimulating the cells that are tobe observed. Stimulation may be direct or indirect (e.g., opticalstimulation of an optical actuator or stimulating an upstream cell insynaptic communication with the cell(s) to be observed). Stimulation maybe optical, electrical, chemical, or by any other suitable method.Stimulation may involve any pattern of a stimulation including, forexample, regular, periodic pulses, single pulses, irregular patterns, orany suitable pattern. Methods may include varying optical stimulationpatterns in space or time to highlight particular aspects of cellularfunction. For example, a pulse pattern may have an increasing frequency.In certain embodiments, imaging includes stimulating a neuron thatexpresses an optical actuator using pulses of light.

A neuron expressing an Optopatch construct may be exposed to whole-fieldillumination with pulses of blue light (10 ms, 25 mW/cm) to stimulateCheRiff, and simultaneous constant illumination with red light (800W/cm) to excite fluorescence of QuasAr2. The fluorescence of QuasAr2 maybe imaged at a 1 kHz frame rate. Key parameters include temporalprecision with which single spikes can be elicited and recorded,signal-to-noise ratio (SNR) in fluorescence traces, and long-termstability of the reporter signal. Methods provided herein may be foundto optimize those parameters. Further discussion may be found in Foustet al., 2010, Action potentials initiate in the axon initial segment andpropagate through axon collaterals reliably in cerebellar Purkinjeneurons, J. Neurosci 30:6891-6902; and Popovic et al., 2011, Thespatio-temporal characteristics of action potential initiation in layer5 pyramidal neurons: a voltage imaging study, J. Physiol. 589:4167-4187.

In some embodiments, measurements are made using a low-magnificationmicroscope that images a 1.2×3.3 mm field of view with 3 μm spatialresolution and 2 ms temporal resolution. In other embodiments,measurements are made using a high-magnification microscope that imagesa 100 μm field of view with 0.8 μm spatial resolution and 1 ms temporalresolution. A suitable instrument is an inverted fluorescencemicroscope, similar to the one described in the Supplementary Materialto Kralj et al., 2012, Optical recording of action potentials inmammalian neurons using a microbial rhodopsin, Nat. Methods 9:90-95.Briefly, illumination from a red laser 640 nm, 140 mW (Coherent Obis637-140 LX), is expanded and focused onto the back-focal plane of a 60×oil immersion objective, numerical aperture 1.45 (Olympus 1-U2B616).

FIG. 5 gives a functional diagram of components of an optical imagingapparatus 501 according to certain embodiments. A 488 nm blue laser beamis modulated in intensity by an acousto-optic modulator (not shown), andthen reflected off a digital micromirror device (DMD) 505. The DMDimparted a spatial pattern on the blue laser beam (used for CheRiffexcitation) on its way into the microscope. The micromirrors werere-imaged onto the sample 509, leading to an arbitrary user-definedspatiotemporal pattern of illumination at the sample. Simultaneouswhole-field illumination with 640 nm red light excites fluorescence ofthe QuasAr reporter.

With the inverted fluorescence microscope, illumination from a bluelaser 488 nm 50 mW (Omicron PhoxX) is sent through an acousto-opticmodulator (AOM; Gooch and Housego 48058-2.5-.55-5W) for rapid controlover the blue intensity. The beam is then expanded and modulated by DMD505 with 608×684 pixels (Texas Instruments LightCrafter). The DMD iscontrolled via custom software (Matlab) through a TCP/IP protocol. TheDMD chip is re-imaged through the objective onto the sample, with theblue and red beams merging via a dichroic mirror. Each pixel of the DMDcorresponds to 0.65 μm in the sample plane. A 532 nm laser is combinedwith the red and blue beams for imaging of mOrange2. Software is writtento map DMD coordinates to camera coordinates, enabling precise opticaltargeting of any point in the sample.

To achieve precise optical stimulation of user-defined regions of aneuron, pixels on DMD 505 are mapped to pixels on the camera. The DMDprojects an array of dots of known dimensions onto the sample. Thecamera acquires an image of the fluorescence. Custom software locatesthe centers of the dots in the image, and creates an affinetransformation to map DMD coordinates onto camera pixel coordinates.

A dual-band dichroic filter (Chroma zt532/635rpc) separates reporter(e.g., Arch) from excitation light. A 531/40 nm bandpass filter (SemrockFF01-531/40-25) may be used for eGFP imaging; a 710/100 nm bandpassfilter (Chroma, HHQ710/100) for Arch imaging; and a quad-band emissionfilter (Chroma ZET405/488/532/642m) for mOrange2 imaging andpre-measurement calibrations. A variable-zoom camera lens (Sigma 18-200mm f/3.5-6.3 II DC) is used to image the sample onto an EMCCD camera(Andor iXon+DU-860), with 128×128 pixels. Images may be first acquiredat full resolution (128×128 pixels). Data is then acquired with 2×2pixel binning to achieve a frame rate of 1,000 frames/s. For runs withinfrequent stimulation (once every 5 s), the red illumination is only onfrom 1 s before stimulation to 50 ms after stimulation to minimizephotobleaching. Cumulative red light exposure may be limited to <5 min.per neuron.

Low magnification wide-field imaging is performed with a custommicroscope system based around a 2x , NA 0.5 objective (Olympus MVX-2).Illumination is provided by six lasers 640 nm, 500 mW (Dragon Lasers635M500), combined in three groups of two. Illumination is coupled intothe sample using a custom fused silica prism, without passing throughthe objective. Fluorescence is collected by the objective, passedthrough an emission filter, and imaged onto a scientific CMOS camera(Hamamatsu Orca Flash 4.0). Blue illumination for channelrhodopsinstimulation is provided by a 473 nm, 1 W laser (Dragon Lasers),modulated in intensity by an AOM and spatially by a DMD (Digital LightInnovations DLi4130-ALP HS). The DMD is re-imaged onto the sample viathe 2x objective. During a run, neurons may be imaged using wide-fieldillumination at 488 nm and eGFP fluorescence. A user may select regionsof interest on the image of the neuron, and specify a time course forthe illumination in each region. The software maps the user-selectedpixels onto DMD coordinates and delivers the illumination instructionsto the DMD.

The inverted fluorescence micro-imaging system records optically fromnumerous (e.g., 50) expressing cells or cell clusters in a single fieldof view. The system may be used to characterize optically evoked firingpatterns and AP waveforms in neurons expressing an Optopatch construct.Each field of view is exposed to whole-field pulses of blue light toevoke activity (e.g., 0.5 s, repeated every 6 s, nine intensitiesincreasing from 0 to 10 mW/cm). Reporter fluorescence such as fromQuasAr may be simultaneously monitored with whole-field excitation at640 nm, 100 W/cm.

FIG. 6 illustrates a pulse sequence of red and blue light used to recordaction potentials under increasing optical stimulation. In someembodiments, neurons are imaged on a high resolution microscope with 640nm laser (600 W/cm) for voltage imaging. In certain embodiments, neuronsare imaged on a high resolution microscope with 640 nm laser (600 W/cm)for voltage imaging and excited with a 488 nm laser (20-200 mW/cm).Distinct firing patterns can be observed (e.g., fast adapting andslow-adapting spike trains). System measurements can detect rareelectrophysiological phenotypes that might be missed in a manual patchclamp measurement. Specifically, the cells' response to stimulation(e.g., optical actuation) may be observed. Instruments suitable for useor modification for use with methods of the invention are discussed inU.S. Pub. 2013/0170026 to Cohen, incorporated by reference.

Using the described methods, populations of cells may be measured. Forexample, both diseased and corrected (e.g., by zinc finger domains)motor neurons may be measured. A cell's characteristic signature such asa neural response as revealed by a spike train may be observed.

5b. Extracting Fluorescence from Movies

Fluorescence values are extracted from raw movies by any suitablemethod. One method uses the maximum likelihood pixel weighting algorithmdescribed in Kralj et al., 2012, Optical recording of action potentialsin mammalian neurons using a microbial rhodopsin, Nat Methods 9:90-95.Briefly, the fluorescence at each pixel is correlated with thewhole-field average fluorescence. Pixels that showed strongercorrelation to the mean are preferentially weighted. This algorithmautomatically finds the pixels carrying the most information, andde-emphasizes background pixels.

In movies containing multiple cells, fluorescence from each cell isextracted via methods known in the art such as Mukamel, Eran A., AxelNimmerjahn, and Mark J. Schnitzer. “Automated analysis of cellularsignals from large-scale calcium imaging data.” Neuron 63.6 (2009):747-760, or Maruyama, Ryuichi, et al. “Detecting cells usingnon-negative matrix factorization on calcium imaging data.” NeuralNetworks 55 (2014): 11-19. These methods use the spatial and temporalcorrelation properties of action potential firing events to identifyclusters of pixels whose intensities co-vary, and associate suchclusters with individual cells.

Alternatively, a user defines a region comprising the cell body andadjacent neurites, and calculates fluorescence from the unweighted meanof pixel values within this region. With the improved trafficking of theQuasAr mutants compared to Arch, these two approaches give similarresults. In low-magnification images, direct averaging and the maximumlikelihood pixel weighting approaches may be found to provide optimumsignal-to-noise ratios.

6. Signal Processing

6a. Signal Processing with Independent Component Analysis to AssociateSignals with Cells

An image or movie may contain multiple cells in any given field of view,frame, or image. In images containing multiple neurons, the segmentationis performed semi-automatically using an independent components analysis(ICA) based approach modified from that of Mukamel, et al., 2009,Automated analysis of cellular signals from large-scale calcium imagingdata, Neuron 63:747-760. The ICA analysis can isolate the image signalof an individual cell from within an image.

FIG. 7-FIG. 10 illustrate the isolation of individual cells in a fieldof view. Individual cells are isolated in a field of view using anindependent component analysis.

FIG. 7 shows an image that contains five neurons whose images overlapwith each other. The fluorescence signal at each pixel is an admixtureof the signals from each of the neurons underlying that pixel.

As shown in FIG. 8, the statistical technique of independent componentsanalysis finds clusters of pixels whose intensity is correlated within acluster, and maximally statistically independent between clusters. Theseclusters correspond to images of individual cells comprising theaggregate image of FIG. 7.

From the pseudo-inverse of the set of images shown in FIG. 8 arecalculated spatial filters with which to extract the fluorescenceintensity time-traces for each cell. Filters are created by setting allpixel weights to zero, except for those in one of the image segments.These pixels are assigned the same weight they had in the original ICAspatial filter.

In FIG. 9, by applying the segmented spatial filters to the movie data,the ICA time course has been broken into distinct contributions fromeach cell. Segmentation may reveal that the activities of the cells arestrongly correlated, as expected for cells found together by ICA. Inthis case, the spike trains from the image segments are similar but showa progress over time as the cells signal one another.

FIG. 10 shows the individual filters used to map (and color code)individual cells from the original image.

6b. Signal Processing Via Sub-Nyquist Action Potential Timing (SNAPT)

For individual cells, action potentials can be identified as spiketrains represented by the timing at which an interpolated actionpotential crosses a threshold at each pixel in the image. Identifyingthe spike train may be aided by first processing the data to removenoise, normalize signals, improve SNR, other pre-processing steps, orcombinations thereof. Action potential signals may first be processed byremoving photobleaching, subtracting a median filtered trace, andisolating data above a noise threshold. The spike train may then beidentified using an algorithm based on sub-Nyquist action potentialtiming such as an algorithm based on the interpolation approach ofFoust, et al., 2010, Action potentials initiate in the axon initialsegment and propagate through axon collaterals reliably in cerebellarPurkinje neurons. J. Neurosci 30, 6891-6902 and Popovic et al, 2011, Thespatio-temporal characteristics of action potential initiation in layer5 pyramidal neurons: a voltage imaging study. J. Physiol. 589,4167-4187.

A sub-Nyquist action potential timing (SNAPT) algorithm highlightssubcellular timing differences in AP initiation. For example, thealgorithm may be applied for neurons expressing Optopatch1, containing avoltage reporter such as QuasAr1. Either the soma or a small dendriticregion is stimulated. The timing and location of the ensuing APs ismonitored.

FIG. 11 shows a patterned optical excitation being used to induce actionpotentials. Movies of individual action potentials are acquired (e.g.,at 1,000 frames/s), temporally registered, and averaged.

The first step in the temporal registration of spike movies is todetermine the spike times. Determination of spike times is performediteratively. A simple threshold-and-maximum procedure is applied to F(t)to determine approximate spike times, {T0}. Waveforms in a brief windowbracketing each spike are averaged together to produce a preliminaryspike kernel K0(t). A cross-correlation of K0(t) with the originalintensity trace F(t) is calculated. Whereas the timing of maxima in F(t)is subject to errors from single-frame noise, the peaks in thecross-correlation, located at times {T}, are a robust measure of spiketiming. A movie showing the mean AP propagation may be constructed byaveraging movies in brief windows bracketing spike times {T}. Typically100-300 APs are included in this average. The AP movie has highsignal-to-noise ratio. A reference movie of an action potential is thuscreated by averaging the temporally registered movies (e.g., hundreds ofmovies) of single APs. Each frame of the movie is then corrected bydividing by this baseline.

Spatial and temporal linear filters may further decrease the noise in APmovie. A spatial filter may include convolution with a Gaussian kernel,typically with a standard deviation of 1 pixel. A temporal filter may bebased upon Principal Components Analysis (PCA) of the set ofsingle-pixel time traces. The time trace at each pixel is expressed inthe basis of PCA eigenvectors. Typically the first 5 eigenvectors aresufficient to account for >99% of the pixel-to-pixel variability in APwaveforms, and thus the PCA Eigen-decomposition is truncated after 5terms. The remaining eigenvectors represented uncorrelated shot noise.

FIG. 12 shows eigenvectors resulting from a principal component analysis(PCA) smoothing operation performed to address noise. Photobleaching orother such non-specific background fluorescence may be addressed bythese means.

FIG. 13 shows a relation between cumulative variance and eigenvectornumber. FIG. 14 gives a comparison of action potential waveforms beforeand after the spatial and PCA smoothing operations.

A smoothly varying spline function may be interpolated between thediscretely sampled fluorescence measurements at each pixel in thissmoothed reference AP movie. The timing at each pixel with which theinterpolated AP crosses a user-selected threshold may be inferred withsub-exposure precision. The user sets a threshold depolarization totrack (represented as a fraction of the maximum fluorescence transient),and a sign for dV/dt (indicating rising or falling edge. The filtereddata is fit with a quadratic spline interpolation and the time ofthreshold crossing is calculated for each pixel.

FIG. 15 shows an action potential timing map. The timing map may beconverted into a high temporal resolution SNAPT movie by highlightingeach pixel in a Gaussian time course centered on the local AP timing.The SNAPT fits are converted into movies showing AP propagation asfollows. Each pixel is kept dark except for a brief flash timed tocoincide with the timing of the user-selected AP feature at that pixel.The flash followed a Gaussian time-course, with amplitude equal to thelocal AP amplitude, and duration equal to the cell-average timeresolution, σ. Frame times in the SNAPT movies are selected to be˜2-fold shorter than a. Converting the timing map into a SNAPT movie isfor visualization; propagation information is in the timing map.

FIG. 16 shows the accuracy of timing extracted by the SNAPT algorithmfor voltage at a soma via comparison to a simultaneous patch clamprecording. FIG. 17 gives an image of eGFP fluorescence, indicatingCheRiff distribution.

FIG. 18 presents frames from a SNAPT movie formed by mapping the timinginformation from FIG. 16 onto a high spatial resolution image from FIG.17. In FIG. 17, the white arrows mark the zone of action potentialinitiation in the presumed axon initial segment (AIS). FIGS. 16-18demonstrate that methods of the invention can provide high resolutionspatial and temporal signatures of cells expressing an optical reporterof neural activity.

After acquiring Optopatch data, cells may be fixed and stained forankyrin-G, a marker of the AIS. Correlation of the SNAPT movies with theimmunostaining images establish that the AP initiated at the distal endof the AIS. The SNAPT technique does not rely on an assumed AP waveform;it is compatible with APs that change shape within or between cells.

The SNAPT movies show AP initiation from the soma in single neurites inmeasured cells. The described methods are useful to reveal latenciesbetween AP initiation at the AIS and arrival in the soma of 320±220 μs,where AP timing is measured at 50% maximum depolarization on the risingedge. Thus Optopatch can resolve functionally significant subcellulardetails of AP propagation. Discussion of signal processing may be foundin Mattis et al., 2011, Principles for applying optogenetic toolsderived from direct comparative analysis of microbial opsins, Nat. Meth.9:159-172; and Mukamel et al., 2009, Automated analysis of cellularsignals from large-scale calcium imaging data, Neuron 63(6):747-760.

Methods of the invention are used to obtain a signature from theobserved cell or cells tending to characterize a physiological parameterof the cell. The measured signature can include any suitableelectrophysiology parameter such as, for example, activity at baseline,activity under different stimulus strengths, tonic vs. phasic firingpatterns, changes in AP waveform, others, or a combination thereof.Measurements can include different modalities, stimulation protocols, oranalysis protocols. Exemplarily modalities for measurement includevoltage, calcium, ATP, or combinations thereof. Exemplary stimulationprotocols can be employed to measure excitability, to measure synaptictransmission, to test the response to modulatory chemicals, others, andcombinations thereof. Methods of invention may employ various analysisprotocols to measure: spike frequency under different stimulus types,action potential waveform, spiking patterns, resting potential, spikepeak amplitude, others, or combinations thereof.

In certain embodiments, the imaging methods are applied to obtain asignature mean probability of spike for cells from the patient and mayalso be used to obtain a signature from a control line of cells such asa wild-type control (which may be produced by genome editing asdescribed above so that the control and the wild-type are isogenic butfor a single site). The observed signature can be compared to a controlsignature and a difference between the observed signature and theexpected signature corresponds to a positive diagnosis of the condition.

FIG. 19 shows a mean probability of spike of wild-type (WT) and mutant(SOD1) cells. Cellular excitability was measured by probability ofspiking during each blue light stimulation, and during no stimulation(spontaneous firing).

7. Diagnosis

FIG. 19 illustrates an output from measuring action potentials in cellsaffected by a mutation and control cells isogenic but for the mutation.In the illustrated example, a patient known to have SOD1A4V is studiedand the bottom trace is obtained from cells of that patient's genotype.The top trace labeled “WT” refers to cells from that patient that wereedited to be SOD1V4A and thus wild-type at the locus of the patient'sknown mutation but otherwise to provide the genetic context present inthe patient. A clinician may diagnosis a neurodegenerative disease basedon a signature spike train manifest by the patient's cells. Here, adifference between the signature observed in the patient's cells and thecontrol signature may be correlated to a positive diagnosis of aneurodegenerative disease.

Any suitable method of correlating the patient's signature to adiagnosis may be used. For example, in some embodiments, visualinspection of a signature may be used. In certain embodiments, acomputer system may be used to automatically evaluate that an observedsignature of the test cells satisfies predetermined criteria for adiagnosis. Any suitable criteria can be used. For example, a computersystem may integrate under the spike train for both the test cells andthe control cells over a range of time of at least a few thousand ms andcompare a difference between the results. Any suitable differencebetween the observed and expected signals can be used, for example, thedifference may include a modified probability of a voltage spike inresponse to the stimulation of the cell relative to a control. Incertain embodiments (e.g., FIG. 19) the difference between the observedsignal and the expected signal comprises a decreased probability of avoltage spike in response to the stimulation of the cell relative to acontrol and an increased probability of a voltage spike during periodsof no stimulation of the cell relative to a control. In one embodiment,systems and methods of the invention detect a decreased probability of avoltage spike in response to the stimulation of the cell relative to acontrol.

To give one example, a difference of at least 5% can be reported asindicative of an increased risk or diagnosis of a condition. In anotherexample, a computer system can analyze a probability of spike at acertain time point (e.g., 5500 ms) and look for a statisticallysignificant difference. In another example, a computer system can beprogrammed to first identify a maximal point in the WT spike train(control signature) and then compare a probability at that point in thecontrol signature to a probability in the patient's test signature atthe same point and look for a reportable difference (e.g., at least 5%different). One of skill in the art will recognize that any suitablecriterion can be used in the comparison of the test signature to thecontrol signature. In certain embodiments, a computer system is trainedby machine learning (e.g., numerous instances of known healthy and knowndiseased are input and a computer system measures an average differencebetween those or an average signature pattern of a disease signature).Where the computer system stores a signature pattern for a diseasephenotype, a diagnosis is supported when the computer system finds amatch between the test signature and the control signature (e.g., <5%different or less than 1% different at some point or as integrated overa distance). While obtaining a control signature from a genome-editedcell line from the patient has been discussed, one of skill in the artwill recognize that the control signature can be a template ordocumented control signature stored in computer system of the invention.

In certain embodiments, observation of a signature from a cell is usedin a diagnosis strategy in which the observed signature phenotypecontributes to arriving at a final diagnosis. For example, with certaindisease of the nervous system such as ALS, different neuron types may beaffected differently. In some embodiments, a diagnostic method includescomparing different neuron types from the same patient to diagnose asub-type specific disease.

8. Additional Methods

Methods of the invention may include the use of tool/test compounds orother interventional tools applied to the observed cell or cells.Application of test compounds can reveal effects of those compounds oncellular electrophysiology. Use of a tool compounds can achieve greaterspecificity in diagnosis or for determining disease mechanisms, e.g. byblocking certain ion channels. By quantifying the impact of thecompound, one can quantify the level of that channel in the cell.

With a tool or test compound, a cell may be caused to express an opticalreporter of neural or electrical activity and may also be exposed to acompound such as a drug. A signature of the cell can be observed before,during, or after testing the compound. Any combination of differentcells and cell types can be exposed to one or any combination ofcompounds, including different test compound controls. Multi-wellplates, multi-locus spotting on slides, or other multi-compartment labtools can be used to cross-test any combination of compounds and celltypes.

In certain embodiments, tool compounds are added to cells and theireffect on the cells is observed to distinguish possible diseases orcauses or mechanisms of diseases. For example, where two or more cellsin synaptic connection with one another are observed, extrinsicstimulation of an upstream cell should manifest as an action potentialin a downstream cell. A compound that is known to inhibitneurotransmitter reuptake may be revealed to work on only certain neuralsubtypes thus indicating a specific disease pattern.

In some embodiments, methods of the invention are used to detect,measure, or evaluate synaptic transmission. A signature may be observedfor a cell other than the cell to which direct stimulation was applied.In fact, using the signal processing algorithms discussed herein,synaptic transmission among a plurality of cells may be detected thusrevealing patterns of neural connection. Establishing an assay thatsuccessfully detects firing of a downstream neuron upon stimulation ofan upstream neuron can reveal, where the subject cell to be observedfails to fire upon stimulation of an upstream neuron, a disease orcondition characterized by a failure of synaptic transmission.

Test compounds can be evaluated as candidate therapies to determinesuitability of a treatment prior to application to patient. E.g. one cantest epilepsy drugs to find the one that reverts the firing pattern backto wild-type. In some embodiments, the invention provides systems andmethods for identifying possible therapies for a patient by testingcompounds, which systems and methods may be employed as personalizedmedicine. Due to the nature of the assays described herein, it may bepossible to evaluate the effects of candidate therapeutic compounds on aper-patient basis thus providing a tool for truly personalized medicine.For example, an assay as described herein may reveal that a patientsuffering from a certain disease has neurons or neural subtypes thatexhibit a disease-type physiological phenotype under the assaysdescribed herein. One or a number of different compounds may be appliedto those neurons or neural subtypes. Cells that are exposed to one ofthose different compounds (or a combination of compounds) may exhibit achange in physiological phenotype from disease-type to normal. Thecompound or combination of compounds that affects the change inphenotype from disease-type to normal is thus identified as a candidatetreatment compound for that patient.

Embodiments of the invention provide modified neurons and methods forthe optical evaluation of diseases autism affecting electrically activecells such as neurons. In some embodiments, neurons and methods of theinvention are used to evaluate a condition known to be associated with agenetic variant, or mutation.

Embodiments of the invention provide modified neurons and methods forthe optical evaluation of diseases epilepsy affecting electricallyactive cells such as neurons. In some embodiments, neurons and methodsof the invention are used to evaluate a condition known to be associatedwith a genetic variant, or mutation.

Embodiments of the invention relate to Alzheimer's. Alzheimer's diseaseis a neurodegenerative disease of uncertain cause (although mutations incertain genes have been linked to the disorder) and is one of the mostcommon forms of dementia. Alzheimer's disease is discussed in Israel etal., 2012, Probing sporadic and familial Alzheimer's disease usinginduced pluripotent stem cells, Nature 482(7384):216-20; Muratore etal., 2014, The familial Alzheimer's disease APPV717I mutation alters APPprocessing and tau expression in iPSC-derived neurons, Human MolecularGenetics, in press; Kondo et al., 2013, Modeling Alzheimer's diseasewith iPSCs reveals stress phenotypes associated with intracellular Abetaand differential drug responsiveness, Cell Stem Cell 12(4):487-496; andShi et al., 2012, A human stem cell model of early Alzheimer's diseasepathology in Down syndrome, Sci Transl Med 4(124):124ra129, the contentsof each of which are incorporated by reference. Systems and methods ofthe invention may be used to evaluate compounds such as correctormolecules for their effect on Alzheimer's affected cells.

The use of stem cell technology provides a clinically-relevant cellmodels of Alzheimer's and the use of microbial optogenetic constructsallows for rapid screening or detection of cellular physiologies andphenotypes. Methods of the invention can provide genetically modifiedneurons that can replicate Alzheimer's disease pathology in in vitro andin vivo conditions in order to develop and test Alzheimer disease drugsin human brain cells.

To recapitulate the disease phenotype, the neurons may be exposed toAβ1-42. Additionally, prospective compounds such as antibodies againstepitopes on Aβ may be studied by methods of the invention. For example,the BIIB037 antibody may be exposed to neurons using systems and methodsof the invention. Optogenetic constructs provide for the optical studyof both the toxicity of the Aβ peptide and the neuroprotective effectsof prospective compounds. Thus methods of the invention provide a modelsystem to study Alzheimer's disease pathology. FIG. 1 diagrams a method101 for evaluating a condition according to embodiments of theinvention. This may involve obtaining 107 a cell (e.g., purchasing PSCsand converting to neurons; biopsy from a person suspected of having thecondition; etc.). Genome editing techniques (e.g., use of transcriptionactivator-like effector nucleases (TALENs), the CRISPR/Cas system, zincfinger domains) may be used to create a control cell that is isogenicbut-for a variant of interest. The cell and the control are convertedinto an electrically excitable cell such as a neuron. The cell may beconverted to a specific neural subtype (e.g., motor neuron). The cellsare caused to express 113 an optical reporter of neural activity. Forexample, the cell may be transformed with a vector comprising anoptogenetic reporter and the cell may also be caused to express anoptogenetic actuator (aka activator) by transformation. Optionally, acontrol cell may be obtained, e.g., by taking another sample, by genomeediting, or by other suitable techniques. Using microscopy andanalytical methods described herein, the cells are observed andspecifically, the cells' response to stimulation 119 (e.g., optical,synaptic, chemical, or electrical actuation) may be observed. A cell'scharacteristic signature such as a neural response as revealed by aspike train may be observed 123. The observed signature is compared to acontrol signature and a difference (or match) between the observedsignature and the control signature corresponds to a positive diagnosisof the condition.

In one exemplary embodiment discussed herein, neurons of the inventioncomprise a genome associated with Alzheimer's disease and are used foroptical evaluation of Alzheimer's disease development, progression,and/or treatments.

In certain embodiments, the invention provides modified neurons andmethods for the optical evaluation of diseases such as tuberoussclerosis affecting electrically active cells such as neurons. In someembodiments, neurons and methods of the invention are used to evaluate acondition known to be associated with a genetic variant, or mutation.Neurons of the invention may be human derived or derived from anotheranimal and may be cultured in vitro or may be modified within a livinganimal, such as a mouse, in order to provide an in vivo disease modelwith optical actuators and reporters of neuronal action potential. Inone exemplary embodiment discussed herein, neurons of the inventioncomprise a genome associated with tuberous sclerosis and are used foroptical evaluation of tuberous sclerosis development, progression,and/or treatments.

In certain aspects, the invention relates to optogenetic methods forrobust, biologically relevant assays with sufficient capacity for highthroughput screening of ion channel modulators. Ion channels aretherapeutic targets and may be modulated by a range of drugs. Iontransport mediated by ion channels is important in many fundamentalphysiological processes in the heart and the nervous system as well asfor fluid secretion in the lung, GI tract and kidney, and otherprocesses such as hormone secretion, the immune response, bonere-modeling and tumor cell proliferation. The physiological importanceof ion channels is underlined by their involvement in a wide range ofpathologies spanning all major therapeutic areas. For example, over 55different inherited ion channel diseases, known as “channelopathies,”have now been identified across cardiovascular, neuronal, neuromuscular,musculoskeletal, metabolic, and respiratory systems. Ion channels aretypically multimeric, transmembrane proteins having separatepore-forming and accessory subunits (Ashcroft, 2006, Nature 440:440-7).Ion channels are often classified according to gating mechanism:voltage-gated channels are regulated by changes in the electricalpotential difference in membrane potential whereas ligand- andsensory-gated channels respond to changes ligands and to mechanical orthermal stimuli, respectively.

High throughput screening of large chemical libraries generally mayinclude cloning of the target protein which is abundantly expressed in astable cell line in a form that closely resembles its native correlates.For ion channels this involves efficient expression, localization, andorientation of an appropriate combination of subunits.

Methods of the invention provide an optical alternative to patch clampelectrophysiology. Methods and the optogenetic constructs of theinvention may be used for high throughput screening (HTS) of ionchannels.

9. Disease Models

The invention provides methods for screening, detecting, andcharacterizing compounds in high-throughput cellular assays of cellsexpressing optogenetic proteins that initiate and report electricalactivity in cells using light. Thus the invention provides high-capacitymethods for primary screening of chemical libraries. Thesehigh-throughput assays provide robust electrophysiological measurementsof cells without requiring patch clamp techniques. Since the describedoptogenetic constructs and pluripotent stem cell (PSC)-derived cellsoperate to provide the precision, temporal resolution, and voltagecontrol required for monitoring effects of compounds, assays of theinvention are compatible with primary screening and drug discovery. Forthe assays, a target protein may be cloned and expressed in a stablecell line of the invention. Thus the invention provides robust,biologically relevant assays with sufficient capacity for highthroughput screening of compounds.

Aspects of the invention provide a method for determining an effect of acompound a neurological condition. The method includes presenting acompound to a sample comprising a plurality of neurons, wherein at leastone of the plurality of neurons expresses an optical reporter ofmembrane electrical potential, and receiving—via a microscopy system—anoptical signal generated by the optical reporter in response to opticalstimulation of a light gated ion channel in the sample followingpresentation of said compound. The compound is identified as a candidatefor treatment of the neurological condition based on said opticalsignal. The light gated ion channel may include an algalchannelrhodopsin being expressed by a second neuron in synapticcommunication with the at least one of the plurality of neurons. Thelight gated ion channel may include an algal channelrhodopsin beingexpressed by the at least one of the plurality of neurons. The opticalreporter of membrane potential may include a microbial rhodopsin (e.g.,with between 1 and 10 amino acid substitutions relative to a wild typeform of the microbial rhodopsin). In some embodiments, the at least oneof the plurality of neurons also expresses a genetically-encodedindicator of intracellular calcium level. The received optical signalmay include a signal from the genetically-encoded indicator ofintracellular calcium level. The neurological condition may be one ofautism, epilepsy, Alzheimer's, amyotrophic lateral sclerosis, andtuberous sclerosis.

i. Autism

In certain embodiments, neurons and methods of the invention may be usedto create disease models for in vitro investigation ofneurodevelopmental disorders such as autism. Neurons may be derived fromiPSCs taken from individuals suffering from the neurodevelopmentaldisorder or may be derived through genome editing by incorporatinggenotype associated with the neurodevelopmental disorder. In certaininstances a test mutation, suspected of being associated with aneurodevelopmental disorder, may be incorporated into a neuron throughgenome editing and the resulting modified neuron may be observed forsigns of disease to evaluate the test mutation for links to the disease.

In some embodiments, cell neuronal models of a disease, such as autismmay be chosen based on the exhibition of neuronal phenotypes associatedwith autism, such as neurons with reduced expression of SHANK3 proteincompared to a disease-free neuron, decreased synaptic function comparedto a disease-free neuron, reduced number and increased length ofdendritic spines compared to a disease-free neuron, and reducedthickness and length of postsynaptic density compared to a disease-freeneuron. See Zoghbi, et al., 2012, Synaptic Dysfunction inNeurodevelopmental Disorders Associated with Autism and IntellectualDisabilities, Cold Spring Harb Perspect Biol. 4(3), J Neurol Sci.217(1):47-54; incorporated by reference. Neuronal models of a diseasesuch as autism may be selected based on genotypic characteristics suchas a mutation to one or more of the following genes: SHANK3 (ProSAP2),CDH9, CDH10, MAPK3, SERT (SLC6A4), CACNA1G, GABRB3, GABRA4, EN2, the3q25-27 locus, SLC25Al2, HOXA1, HOXA2, PRKCB1, MECP2, UBE3A, NLGN3, MET,CNTNAP2, FOXP2, GSTP1, PRL, PRLR, and OXTR.

In certain aspects, for example where modelled disease arenon-monogenic, complex etiology and/or late onset, neurons of theinvention may be cultured for extended periods, such as 1 month, 2months, 3 months, 4 months or longer in order to simulate aging. SeeSanchez-Danes, et al., 2012, Disease-specific phenotypes in dopamineneurons from human iPS-based models of genetic and sporadic Parkinson'sdisease, EMBO Mol Med, 4: 380-395, the contents of which areincorporated by reference. Cells of the invention may be transformedwith optical reporters of membrane potential, reporters of intracellularcalcium levels, light-gated ion channels, or a combination thereof.Cells may be monitored over time by inducing and observing actionpotentials and changes in intracellular calcium levels during diseaseprogression in order to examine the neuronal effects of the subjectcondition, such as autism. Subject cells of the disease model may alsobe monitored pre and post application of various therapies in order toevaluate their effectiveness.

ii. Epilepsy

In certain embodiments, neurons and methods of the invention may be usedto create disease models for in vitro investigation of neurologicaldisorders such as epilepsy. Neurons may be derived from iPSCs taken fromindividuals suffering from the neurological disorder or may be derivedthrough genome editing by incorporating a genotype associated with theneurological disorder. Disease models of the invention may beparticularly useful in studying action potential generation andpropagation and ion channel function before, during, and after anepileptic seizure. In certain instances a test mutation, suspected ofbeing associated with a neurological disorder, may be incorporated intoa neuron through genome editing and the resulting modified neuron may beobserved for signs of disease to evaluate the test mutation for links tothe disease.

In some embodiments, cell neuronal models of a disease, such as epilepsyor Dravet syndrome, may be chosen based on the exhibition of neuronalphenotypes associated with the disease, such as neurons with diminishedvoltage-gated sodium channel function compared to disease-free neuronsor hyperexcitability. See Kearney, 2014, The More, the Better: ModelingDravet Syndrome With Induced Pluripotent Stem Cell-Derived Neurons,Epilepsy Curr. 14(1): 33-34; incorporated by reference. Neuronal modelsof a disease such as epilepsy or Dravet syndrome may be selected basedon genotypic characteristics such as a mutation to one or more of thefollowing genes: SCN1A, WWOX, PRRT2, KCNC1, STX1B, CARS2, STXB1, KCNQ2,CDKL5, ARX, SPTAN, BRAT1, KCNQ3, SCN2A, GABA receptors, NIPA2, CDKL5,PCDH19, and NAV1.1.

In certain aspects, for example where modelled disease arenon-monogenic, complex etiology and/or late onset, neurons of theinvention may be cultured for extended periods, such as 1 month, 2months, 3 months, 4 months or longer in order to simulate aging. SeeSanchez-Danes, et al., 2012, Disease-specific phenotypes in dopamineneurons from human iPS-based models of genetic and sporadic Parkinson'sdisease, EMBO Mol Med, 4: 380-395, the contents of which areincorporated by reference. Cells of the invention may be transformedwith optical reporters of membrane potential, reporters of intracellularcalcium levels, light-gated ion channels, or a combination thereof.Cells may be monitored over time by inducing and observing actionpotentials and changes in intracellular calcium levels during diseaseprogression in order to examine the neuronal effects of the subjectcondition, such as epilepsy. Subject cells of the disease model may alsobe monitored pre and post application of various therapies in order toevaluate their effectiveness.

iii. ALS

In certain embodiments, neurons and methods of the invention may be usedto create disease models for in vitro investigation of neuronal diseasessuch as ALS. Neurons may be derived from iPSCs taken from individualssuffering from the neuronal disease or may be derived through genomeediting by incorporating genotype associated with the neuronal disease.In certain instances a test mutation, suspected of being associated witha neuronal disease, may be incorporated into a neuron through genomeediting and the resulting modified neuron may be observed for signs ofdisease to evaluate the test mutation for links to the disease. In someembodiments, cell neuronal models of a disease, such as ALS disease maybe chosen based on the exhibition of neuronal phenotypes associated withALS, such as motor neurons with Bunina bodies, which are cystatinC-containing inclusions in the cell body; ‘Lewy body-like inclusions’(LBIs), ‘Skein-like inclusions’ (SLIs) inclusions, and/or clear signs ofdegeneration, including very short or absent neurites, vacuolated soma,a fragmented nucleus and cleaved caspase-3. See He, et al., 2004,Expression of peripherin in ubiquinated inclusions of amyotrophiclateral sclerosis, J Neurol Sci. 217(1):47-54; Kawashima, et al., 1998,Skein-like inclusions in the neostriatum from a case of amyotrophiclateral sclerosis with dementia, Acta Neuropathol 96(5):541-5; Okamoto,et al., 1993, Bunina bodies in amyotrophic lateral sclerosisimmunostained with rabbit anti-cystatin C serum, Neurosci Lett.162(1-2):125-8; each of which is incorporated by reference. Neuronalmodels of a disease such as ALS may be selected based on genotypiccharacteristics such as a mutation to one or more of the followinggenes: C9orf72, SOD1, TARDBP, FUS, UBQL2, ALS2, and SETX.

In certain aspects, for example where modelled disease arenon-monogenic, complex etiology and/or late onset, neurons of theinvention may be cultured for extended periods, such as 1 month, 2months, 3 months, 4 months or longer in order to simulate aging. SeeSánchez-Danés, et al. Cells of the invention may be transformed withoptical reporters of membrane potential, reporters of intracellularcalcium levels, light-gated ion channels, or a combination thereof.Cells may be monitored over time by inducing and observing actionpotentials and changes in intracellular calcium levels during diseaseprogression in order to examine the neuronal effects of the subjectcondition, such as ALS. Subject cells of the disease model may also bemonitored pre and post application of various therapies in order toevaluate their effectiveness.

iv. Tuberous Sclerosis

In certain embodiments, neurons and methods of the invention may be usedto create disease models for in vitro investigation of genetic disorderssuch as tuberous sclerosis. Neurons may be derived from iPSCs taken fromindividuals suffering from the neurological disorder or may be derivedthrough genome editing by incorporating genotype associated with theneurological disorder. Disease models of the invention may beparticularly useful in studying action potential generation andpropagation and ion channel function before, during, and after anepileptic seizure. In certain instances a test mutation, suspected ofbeing associated with a neurological disorder, may be incorporated intoa neuron through genome editing and the resulting modified neuron may beobserved for signs of disease to evaluate the test mutation for links tothe disease.

In some embodiments, cell neuronal models of a disease, such as tuberoussclerosis may be chosen based on the exhibition of neuronal phenotypesassociated with tuberous sclerosis, such as enlarged size compared to adisease-free neuron, increased phospho-S6 expression, prominentlysosomes, more microfilaments and microtubules compared to adisease-free neuron, fewer lipofuscin granules compared to adisease-free neuron, and immunoreactivity for TSC2 gene product,tuberin, vimentin or glial fibrillary acidic protein. See Meikle, etal., 2007; Arai, et al., 1999, A comparison of cell phenotypes inhemimegalencephaly and tuberous sclerosis, Acta Neuropathol.98(4):407-13; each of which is incorporated by reference. Neuronalmodels of a disease such as tuberous sclerosis may be selected based ongenotypic characteristics such as a mutation to one or more of thefollowing genes: TSC1 or TSC2.

In certain aspects, for example where modelled disease arenon-monogenic, complex etiology and/or late onset, neurons of theinvention may be cultured for extended periods, such as 1 month, 2months, 3 months, 4 months or longer in order to simulate aging. SeeSanchez-Danes, et al., 2012, Disease-specific phenotypes in dopamineneurons from human iPS-based models of genetic and sporadic Parkinson'sdisease, EMBO Mol Med, 4: 380-395, the contents of which areincorporated by reference. Cells of the invention may be transformedwith optical reporters of membrane potential, reporters of intracellularcalcium levels, light-gated ion channels, or a combination thereof.Cells may be monitored over time by inducing and observing actionpotentials and changes in intracellular calcium levels during diseaseprogression in order to examine the neuronal effects of the subjectcondition, such as tuberous sclerosis. Subject cells of the diseasemodel may also be monitored pre and post application of varioustherapies in order to evaluate their effectiveness.

v. NGN2 Neurons

Aspects of the invention provide cellular disease models in which stemcells may be converted into functional neurons by forced expression of asingle transcription factor and then also caused to express optogeneticreporters or actuators of neural activity. A transcription factor suchas neurogenin-2 (NgN2) or NeurD1 introduced into a pluripotent stem cellby transfection is expressed, causing the cell to differentiate into aneuron. Additionally or separately an optogenetic construct thatincludes an optical reporter of intracellular calcium as well as anoptical actuator or reporter of membrane potential is expressed.

10. Systems of the Invention

FIG. 20 presents a system 1101 useful for performing methods of theinvention. Results from a lab (e.g., transformed, converted patientcells) are loaded into imaging instrument 501. Imaging instrument 501 isoperably coupled to an analysis system 1119, which may be a PC computeror other device that includes a processor 125 coupled to a memory 127. Auser may access system 1101 via PC 1135, which also includes a processor125 coupled to a memory 127. Analytical methods described herein may beperformed by any one or more processor 125 such as may be in analysissystem 1119, PC 1135, or server 1139, which may be provided as part ofsystem 1101. Server 1139 includes a processor 125 coupled to a memory127 and may also include optional storage system 1143. Any of thecomputing device of system 1101 may be communicably coupled to oneanother via network 1131. Any, each, or all of analysis system 1119, PC1135, and server 1139 will generally be a computer. A computer willgenerally include a processor 125 coupled to a memory 127 and at leastone input/output device.

A processor 125 will generally be a silicon chip microprocessor such asone of the ones sold by Intel or AMD.

Memory 127 may refer to any tangible, non-transitory memory or computerreadable medium capable of storing data or instructions, which—whenexecuted by a processor 125—cause components of system 1101 to performmethods described herein.

Typical input/output devices may include one or more of a monitor,keyboard, mouse, pointing device, network card, Wi-Fi card, cellularmodem, modem, disk drive, USB port, others, and combinations thereof.

Generally, network 1131 will include hardware such as switches, routers,hubs, cell towers, satellites, landlines, and other hardware such asmakes up the Internet.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Optical Differentiation of a Motor Neuron Model ofAmyotrophic Lateral Sclerosis (ALS) Arising from a Monogenic Mutation inthe SOD1 Gene (SOD1A4V)

Methods of the invention were employed to evaluate the effects of amutation on a patient's cells in the genetic context of that patient.ALS is a fatal neurodegenerative disease that affects pyramidal neuronsin the motor cortex and lower motor neurons that originate in thebrainstem and spinal cord. See Musaro, 2010, State of the art and thedark side of amyotrophic lateral sclerosis, WJBC 1(5):62-68. Typicalmanifestations include degeneration of motor neurons leading to muscleweakness and atrophy, speech and swallowing disabilities, paralysis, anddeath by respiratory failure. ALS is classified into sporadic orfamilial forms. It is thought that many of the familiar forms are causedby mutations in the Cu/Zn superoxide dismutase-1 (SOD1) protein. Anothergene that may be used is C9orf72 where an incompletely penetrantmutation is sometimes associated with symptoms. The discussion hererelates to SOD1 and one of skill in the art will recognize that thetechniques apply for mutations in other genes such as C9orf72. SOD1converts the toxic mitochondrial by-product superoxide into water orhydrogen peroxide. Evidence suggests SOD1 mutations are gain-of-functionmutations. See Rotunno & Bosco, 2013, An emerging role for misfoldedwild-type SOD1 in sporadic ALS pathogenesis, Front Cell Neurosci 7:a253;and Saccon, et al., 2013, Is SOD1 loss of function involved inamyotrophic lateral sclerosis?, Brain 136:2342-2358. It is known thatother gene defects besides SOD1 mutations can cause ALS. See Pasinelli &Brown, 2006, Molecular biology of amyotrophic lateral sclerosis:insights from genetics, Nat Rev Neurosci 7:710-723; and Blokhuis et al.,2013, Protein aggregation in amyotrophic lateral sclerosis, ActaNeuropathol 125:777-794. Thus mere identification of the presence of asingle mutation may prove inadequate for diagnosing and treating apatient and it may prove valuable to study the phenotypic consequencesof such a mutation with the patient's actual genetic consequence.Contemporary research supports treatment strategies that aim to slowdisease progression by targeting known genes, physiological pathways,and proteins. For more discussion, see Gordon, 2013, Amyotrophic latersclerosis: an update for 2013 clinical features, pathophysiology,management, and therapeutic trials, Aging and Disease 4(5):295-310. Thefollowing protocol documented an effect of SOD1A4V on motor neurons in acell line from a person with an ALS diagnosis known to have SOD1A4V.

(1) Fibroblasts were taken from a patient diagnosed with ALS andconfirmed mutation in SOD1.

(2) Fibroblasts were converted to induced pluripotent stem (iPS) cells.

(3) A second genetically corrected line (Sod1V4A) was generated usingzinc finger domains resulting in two otherwise isogenic lines.

(4) Diseased and corrected iPS cells were differentiated into motorneurons using embryoid bodies.

(5) Differentiated motor neurons were dissociated and plated onto glasscoverslips coated with poly-d-lysine and laminin

(6) Motor neurons were fed with neurobasal medium supplemented with N2,B27, GDNF, BDNF, and CTNF.

(7) After 4 days in culture, neurons were infected with lenti-virusbearing a genetically encoded fluorescent voltage reporter (QuasAr2) andoptical voltage actuator (CheRiff).

(8) Neurons were further matured for 8-10 days post infection.

(9) Neurons were imaged on a high resolution microscope with 640 nmlaser (600 W/cm) for voltage imaging and excited with a 488 nm laser(20-200 mW/cm).

(10) A pulse sequence of red and blue light was used to record actionpotentials under increasing optical stimulation of voltage (FIG. 6).

(11) A population of cells was measured from diseased and correctedmotor neurons.

(12) Individual cells were isolated in a field of view using independentcomponent analysis (FIGS. 7-10).

(13) Action potentials were identified by removing photobleaching,subtracting a median filtered trace, and isolating data above a noisethreshold.

(14) Cellular excitability was measured by probability of spiking duringeach blue light stimulation, and during no stimulation (spontaneousfiring) (FIG. 19).

What is claimed is:
 1. A method for determining an effect of a compoundon a neurological condition, the method comprising: presenting acompound selected for treating a neurological condition to a cell sampleexhibiting a genotype associated with the neurological condition, thecell sample comprising a plurality of neurons, wherein at least one ofthe plurality of neurons expresses an optical reporter of membraneelectrical potential; optically stimulating an optogenetic actuator inthe sample following presentation of the compound, wherein theoptogenetic actuator is a light gated ion channel; generating an opticalsignal in response to the optical stimulation of the light gated ionchannel using the optical reporter; receiving, via a microscopy system,the optical signal generated by the optical reporter; analyzing theoptical signal generated by the optical reporter with respect to acontrol signal obtained from a control cell sample not exhibiting agenotype associated with the neurological condition; and identifying thecompound as a candidate for treatment of the neurological conditionbased on the analysis of said optical signal with respect to the controlsignal.
 2. The method of claim 1, wherein a plurality of samples areexposed to a plurality of different compounds.
 3. The method of claim 1,wherein the light gated ion channel comprises an algal channelrhodopsinbeing expressed by a second neuron in synaptic communication with the atleast one of the plurality of neurons, and the optical reporter ofmembrane potential comprises a microbial rhodopsin with between 1 and 10amino acid substitutions relative to a wild type form of the microbialrhodopsin.
 4. The method of claim 3, wherein the at least one of theplurality of neurons also expresses a genetically-encoded indicator ofintracellular calcium level, and wherein the receive optical signalincludes a signal from the genetically-encoded indicator ofintracellular calcium level, and further wherein the neurologicalcondition is any one of autism, epilepsy, Alzheimer's, amyotrophiclateral sclerosis, or tuberous sclerosis.
 5. The method of claim 1,wherein each of the plurality of neurons is caused to express both theoptical reporter of membrane electrical potential and the light gatedion channel.
 6. The method of claim 5, further comprising transformingthe neurons with a vector that includes a nucleic acid encoding theoptical reporter of membrane electrical potential and the light gatedion channel.
 7. The method of claim 1, wherein the neurologicalcondition is selected from the group consisting of Cockayne syndrome,Down Syndrome, Dravet syndrome, familial dysautonomia, Fragile XSyndrome, Friedreich's ataxia, Gaucher disease, hereditary spasticparaplegias, Machado-Joseph disease, Phelan-McDermid syndrome (PMDS),polyglutamine (polyQ)-encoding CAG repeats, spinal muscular atrophy,Timothy syndrome, Alzheimer's disease, frontotemporal lobardegeneration, Huntington's disease, multiple sclerosis, Parkinson'sdisease, spinal and bulbar muscular atrophy, and amyotrophic lateralsclerosis.
 8. The method of claim 1, further comprising performing thepresenting, optically stimulating, generating, and receiving steps on acontrol sample, wherein the neurons in the cell sample are isogenic withcells in the control sample but for a mutation.
 9. The method of claim1, wherein receiving the optical signal comprises observing a cluster ofcells with a microscope and using a computer to isolate a signalgenerated by the optical reporter from among a plurality of signals fromthe cluster of cells.
 10. The method of claim 9, wherein the computerisolates the signal by performing an independent component analysis andidentifying a spike train produced by the neuron.
 11. The method ofclaim 10, further comprising using the microscopy system to obtain animage of a plurality of clusters of cells.